Pain-relieving compositions and uses therefor

ABSTRACT

Compositions and methods are for inducing, promoting or otherwise facilitating pain relief. More particularly, sub-normovasodilatory doses of nitric oxide donors are used in the therapeutic management of vertebrate animals including humans, for the prevention or alleviation of pain, especially neuropathic pain. In some applications of the method, nitric oxide donors can be administered by any suitable route so as to provide concentrations of NO that are about ½ to 10 −15  times those known to induce vasodilation in normal circulations.

RELATED APPLICATIONS

This application is a Continuation Application of U.S. application Ser.No. 14/466,795, filed Aug. 22, 2014, which is a Continuation Applicationof U.S. application Ser. No. 12/494,183, filed Jun. 29, 2009, now issuedas U.S. Pat. No. 8,822,509 on Sep. 2, 2014, which is aContinuation-in-Part Application of International Application No.PCT/AU2008/000003, filed Jan. 2, 2008, designating the U.S. andpublished in English on Jul. 10, 2008 as WO 2008/080194, which claimsthe benefit of Australian Application No. 2006907305, filed Dec. 29,2006, Australian Application No. 2008903394, filed Jul. 2, 2008,Australian Application No. 2008904197, filed Aug. 15, 2008, andAustralian Application No. 2009901445, filed Apr. 3, 2009, all of whichare incorporated by reference in their entireties.

FIELD OF THE INVENTION

This invention relates generally to compositions and methods forinducing, promoting or otherwise facilitating pain relief. Moreparticularly, the present invention relates to the use ofsub-normovasodilatory doses of nitric oxide donors in the therapeuticmanagement of vertebrate animals including humans, for the prevention oralleviation of pain, especially neuropathic pain. According to someembodiments of the present invention, nitric oxide donors areadministered by any suitable route so as to provide concentrations of NOthat are about ½ to 10⁻¹⁵ of those known to induce vasodilation innormal circulations.

BACKGROUND OF THE INVENTION

Painful diabetic neuropathy (PDN) is a common and debilitatingperipheral nerve complication of diabetes mellitus. By 1 year after theinitial diagnosis of diabetes, 7% of patients report symptoms (e.g.pain, abnormal sensations) with the prevalence rising to 50% by 25 yearsof diabetes diagnosis (Sima and Sugimoto, Diabetologia 42: 773-788 1999;Cameron et al. Diabetologia 44: 1973-88 2001). Patients may present withone or more symptoms including burning sensations, lancinating and deepaching pains, depending upon the extent of nerve injury (Boulton,Diabetes Metab Res Rev 19: S16-21 2003). Numbness, tingling, and asensation of tightness in the extremity are also commonly associatedwith PDN (Boulton, Diabetes Metab Res Rev 19: S16-21 2003). In addition,unpleasant abnormal sensations (dysaethesia), enhanced sensitivity tostimulation (hyperaesthesia), a heightened response to painful stimuli(hyperalgesia) and a distorted sense of touch producing allodynia(innocuous stimuli such as light brushstrokes of the skin produce pain)are all commonly reported by patients with diabetic neuropathy (Boulton,Diabetes Metab Res Rev 19: S16-21 2003).

There are no preventative treatments for PDN (Sima et al. Diabetologica42: 773-788 1999), hence the therapeutic management of the condition isprimarily palliative. This palliative management also represents asignificant therapeutic obstacle, as the most efficient analgesicpharmaceuticals available, the μ-opioid receptor agonists such asmorphine, are reportedly ineffective for the relief of PDN (Attal, ClinJ Pain 16: S118-30 2006). The mechanism underpinning the development ofthis opioid agonist hyposensitivity is unclear, but investigations haveshown that poor glycaemic control can reduce pain tolerance and painthreshold and thus reduce the effectiveness of analgesics such asmorphine (Morley et al. Am J Med 77(1): 79-83 1984). In addition, theremay be diabetes-associated alterations in morphine pharmacokinetics(Courteix et al. J Pharmacol Exp Ther 285(1): 63-70 1998) and/or changesin opioid receptor function (Chen et al. Anesthesiology 97: 1602-16082002).

Although PDN is attributed primarily to poor glycaemic control over aprolonged period, the exact pathogenesis is poorly understood (Sima andSugimoto, Diabetologia 42: 773-788 1999; Feldman et al. Curr Opin Neurol12: 553-63 1999) Presently, there are two broad theories regarding thedevelopment of PDN: the vascular dysfunction theory and the metabolicdysfunction theory.

The vascular dysfunction theory proposes that changes in the bloodsupply to the nerves (the neurovasculature or vasa nervorum) occursecondary to haemodynamic abnormalities (such as accelerated plateletaggregation and increased blood viscosity) (Fusman et al. Acta Diabetol38(3):129-34 2001). In addition, pathological changes in the small bloodvessels of the neurovasculature may occur (such as reduction of theproduction of nitric oxide from the endothelial cells of blood vesselsand acceleration of the reactivity on vasoconstrictive substances)(McAuley et al. Clin Sci (Lond) 99(3): 175-9 2000). These haemodynamicand vascular changes, acting independently or synergistically, arecapable of causing the perineurial ischemia and subsequent endoneurialhypoxia observed in human patients and animal models of diabetes(Cameron et al. Diabetologia 44(11): 1973-88 2001). The end result ofthese abnormalities is nerve damage capable of causing the symptoms andsigns of PDN.

On the other hand, in the metabolic dysfunction theory, the causes ofnerve damage are mediated through the activation of the polyol metabolicpathway and through non-enzymatic protein glycation. These pathwaysinduce mitochondrial and cytosolic NAD⁺/NADH redox imbalances and energydeficiencies in the nerves which can culminate in damage to neural andneurovascular tissues (Obrosova et al. FASEB J 16(1):123-5 2002). Inaddition, these metabolic changes are thought to activate protein kinaseC (PKC) which is capable of heightening pain responses (Kamei et al.Expert Opin Investig Drugs 10(9): 1653-64 2001) and also of producingμ-opioid agonist hyposensitivity (Wang et al. Brain Res 723(1-2): 61-91996). Furthermore, heightened PKC activity is thought to reduce thebinding affinity of μ-opioid receptors for ligands (Ohsawa et al. BrainRes 764:244-8 1998). The consequences of these metabolic abnormalitiesare nerve damage and the development of μ-opioid agonisthyposensitivity, as seen in patients with PDN.

It is likely that neither theory is mutually exclusive and proponents ofboth theories converge in the belief that, downstream of vasculardysfunction or metabolic abnormalities, there is an imbalance in theproduction of vaso-active compounds in the vasa nervorum which leads tohypoxic ischemia of diabetic nerves.

Of all the endogenous vasodilators, nitric oxide is the most potent andhence is a likely candidate for reduced synthesis and consequentdiabetes-induced constrictions in vascular tone. As well as relaxingvascular smooth muscle, it also inhibits the processes of plateletaggregation, mitogenesis and proliferation of cultured vascular smoothmuscle, and leucocyte adherence (Wroblewski et al. Prev Cardiol 3(4):172-177 2000). Nitric oxide is produced by the vascular endothelium by agroup of enzymes called nitric oxide synthases. There are three isoformsof nitric oxide synthase (NOS) named according to their activity or thetissue type in which they were first described. These enzymes allconvert the endogenous substrate, L-arginine, into L-citrulline,producing NO in the process.

Recent studies by the present inventors revealed unexpectedly thatnitric oxide donors such as L-arginine can broadly prevent, attenuateand/or reverse the development of reduced analgesic sensitivity to anopioid receptor agonist such as morphine in neuropathic conditions,including peripheral neuropathic conditions such as PDN (seeInternational Publication No. WO 2003/078437). This finding that nitricoxide donors can restore the analgesic sensitivity of opioid analgesicssuch as morphine in subjects with neuropathic conditions was significantbecause it allowed the use of these analgesics for treating orpreventing pain in conditions, for which they were previously consideredineffective.

SUMMARY OF THE INVENTION

The present inventors have surprisingly discovered that nitric oxide(NO) donors which directly or indirectly generate NO at concentrationsthat are smaller than those known to induce vasodilation in normalcirculations (also referred to herein as sub-normovasodilatory (SNV)concentrations), are effective in producing analgesia in subjects with aneuropathic condition without the need for co-administering opioidanalgesics. Based on this discovery, the present inventors consider thatspecific embodiments of SNV concentrations can broadly range from about½ to about 10⁻¹⁵ of those known to induce vasodilation in normalcirculations.

Accordingly, one aspect of the present invention provides methods forthe treatment or prophylaxis of a neuropathic condition in a subject. Insome embodiments, the neuropathic condition is treated or prevented byadministering to the subject at least one NO donor at a level thatenhances NO and that does not alter normal systemic vascular tone in thesubject. The NO donor may be administered without co-administration ofan opioid analgesic. Thus, in these embodiments, the methods of treatingor preventing the neuropathic condition consist essentially ofadministering the NO donor(s). Suitably, the level of NO is asub-normovasodilatory (SNV) concentration that ranges from about ½ toabout 10⁻¹⁵ of a reference concentration required to induce vasodilationin an anatomical site of a reference subject lacking a vascularcondition, which suitably but not exclusively associates with theneuropathic condition to be treated or prevented. Illustrativeanatomical sites include kidney, skin, skeletal muscle, arm, leg, tailand gastro-intestinal tract.

The NO donor is suitably administered in the form of a compositioncomprising a pharmaceutically acceptable carrier and/or diluent. Thecomposition may be administered by injection, by topical application, orby the buccal, sublingual, rectal or oral routes, includingsustained-release modes of administration, over a period of time and inamounts which are effective for delivering a SNV concentration of NO asbroadly described above. In some embodiments, the NO donor is providedin a sustained release formulation (e.g., transdermal patch), whichdelivers a SNV concentration of NO as broadly described above. In someembodiments, the NO donor is a slow-release NO donor that delivers anSNV concentration of NO as broadly described above.

In accordance with the present invention, SNV concentrations of NO havebeen shown to prevent or attenuate the pain associated with aneuropathic condition. Thus, in another aspect, the invention providesmethods for preventing or attenuating neuropathic pain, especiallyperipheral neuropathic pain, in a subject. In some embodiments,neuropathic pain is prevented or attenuated by administering to thesubject at least one NO donor at a level that enhances NO and that doesnot alter normal systemic vascular tone in the subject, wherein the NOdonor is suitably in the form of a composition comprising apharmaceutically acceptable carrier and/or diluent.

In a further aspect, the present invention contemplates the use of acomposition that consists essentially of at least one NO donor forproducing analgesia in a subject, especially in a subject having aneuropathic condition, which is suitably a peripheral neuropathiccondition such as PDN or a related condition, wherein the compositioncomprises at least one NO donor at a level that enhances NO and thatdoes not alter normal systemic vascular tone in the subject. In someembodiments, the composition excludes an opioid analgesic or is used toproduce analgesia in the absence of co-administering an opioidanalgesic.

In a further aspect of the invention there is provided a compound offormula

wherein R₁ and R₂ are independently selected from hydrogen, alkyl,alkenyl, alkynyl, haloalkyl, —C₀₋₆alkylcycloalkyl,—C₀₋₆alkylcycloalkenyl, —C₀₋₆alkylaryl, —C₀₋₆alkylheterocyclyl,—C₀₋₆alkylheteroaryl, —C₀₋₆alkylCO₂R₃, —C₀₋₆alkylC(O)R₃,—C₀₋₆alkylC(O)NHR₄, —C₀₋₆alkylN(R₄)₂, —C₀₋₆alkylN⁺(R₇)₃, —C₀₋₆alkylOR₅,—C₀₋₆alkylSR₅, —C₀₋₆alkylC(═NR₆)R₃, —C₀₋₆alkylN═NR₅,—C₀₋₆alkylNR₄N(R₄,)₂, —C₀₋₆alkylNR₄C(═NR₄)N(R₄,)₂, —C₀₋₆alkylhalo,—C₀₋₆alkylS(O)R₃, —C₀₋₆alkylSO₂R₃, —CN and —NO₂; or R₁ and R₂ takentogether form an optionally substituted 5 to 8 membered saturated orunsaturated carbocyclic or heterocyclic ring, an aryl ring or aheteroaryl ring;R₃ is selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl,—C₀₋₆alkylcycloalkyl, —C₀₋₆alkylcycloalkenyl, —C₀₋₆alkylaryl,—C₀₋₆alkylheterocyclyl, —C₀₋₆alkylheteroaryl, —C₁₋₆alkylCO₂R₇,—C₀₋₆alkylN(R₄)₂, —C₁₋₆alkylNR₄C(═NR₄)N(R₄,)₂—C₁₋₆alkylOR₇ and—C₁₋₆alkylSR₇;each R₄ is selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl,—C₀₋₆alkylcycloalkyl, —C₀₋₆alkylcycloalkenyl, —C₀₋₆alkylaryl,—C₀₋₆alkylheterocyclyl, —C₀₋₆alkylheteroaryl, —C₀₋₆alkylC(O)R₈,—C₀₋₆alkylC(S)R₈, —C₀₋₆alkylCO₂R₇, —C₀₋₆alkylSO₂R₈ and —C₀₋₆alkylOR₇;R₅ is selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl,—C₀₋₆alkylcycloalkyl, —C₀₋₆alkylcycloalkenyl, —C₀₋₆alkylaryl,—C₀₋₆alkylheterocyclyl, —C₀₋₆alkylheteroaryl, —C₀₋₆alkylC(O)R₇,—C₀₋₆alkylCO₂R₈, —C₀₋₆alkylN(R₇)₂, —C₀₋₆alkylC(O)N(R₇)₂,—C₀₋₆alkylNR₄C(═NR₄)N(R₄,)₂, —C₁₋₆alkylOR₇ and —C₁₋₆alkylSR₇;R₆ is selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl,—C₀₋₆alkylcycloalkyl, —C₀₋₆alkylcycloalkenyl, —C₀₋₆alkylaryl,—C₀₋₆alkylheterocyclyl, —C₀₋₆alkylheteroaryl, —C₀₋₆alkylNHC(O)N(R₇)₂,—C₀₋₆alkylNHC(O)R₇, —C₀₋₆alkylNHSO₂R₇, —C₀₋₆alkylNHCO₂R₇,—C₀₋₆alkylOC(O)R₇, —C₀₋₆alkylC(O)R₇, —CN and —OR₇;each R₇ is selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl,—C₀₋₆alkylcycloalkyl, —C₀₋₆alkylcycloalkenyl, —C₀₋₆alkylaryl,—C₀₋₆alkylheterocyclyl and —C₀₋₆alkylheteroaryl; andR₈ is selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl,—C₀₋₆alkylcycloalkyl, —C₀₋₆alkylcycloalkenyl, —C₀₋₆alkylaryl,—C₀₋₆alkylheterocyclyl, —C₀₋₆alkylheteroaryl and —N(R₇)₂;wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl,heterocyclyl and heteroaryl group is optionally substituted; or apharmaceutically compatible salt thereof; wherein the compound is not4-formyl-3-methyl-1,2,5-oxidiazole-2-oxide.

In a further aspect of the invention, there is provided a compound offormula (II):

X is a covalent bond, —O—, —S— or —N(R₂₃)—;W is a covalent bond, —O—, —S—, —N(R₂₃)— or —C₆H₄—;Y is a covalent bond, —O— or —S—;Z is —NO or —NO₂R₂₀ is hydrogen, —OH, —Oalkyl, —NH₂, —NHalkyl or —N(alkyl)₂;each R₂₁ and R₂₂ is independently selected from hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, halo, hydroxyl, alkyloxy, —CO₂H,—CO₂alkyl, —CONH₂, —CONHalkyl, —CON(alkyl)₂, aryl, heterocyclyl andheteroaryl;R₂₃ is a hydrogen or alkyl;n is 0 or an integer of 1-10; andm is an integer of 1-10.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation showing that tactile (mechanical)allodynia is fully developed by ˜9-10 wks post-STZ administration inadult male Wistar rats.

FIG. 2 is a graphical representation showing mean (±SEM) ΔPWT versustime curves produced by single s.c. bolus doses (1.8-21.4 μmol/Kg) ofmorphine produces dose-dependent (A) antinociception in controlnon-diabetic rats (n=4-9 per dose), and (B) to (D): anti-allodynia inSTZ-diabetic rats at 10 (B; n=4-9 per dose), 14 (C; n=6-10 per dose) and24 (D; n=4-6 per dose) weeks post STZ administration with temporaldevelopment of morphine hyposensitivity across the 24 wk post-STZ studyperiod. By contrast, vehicle (n=3-4) did not produce significantantinociception or anti-allodynia in the hindpaws of either non-diabeticor STZ-diabetic rats respectively.

FIG. 3 is a graphical representation showing mean (±SEM) ΔPWT versustime curves produced by single s.c. bolus doses (8-800 pmol/kg) of thefuroxan NO donor of formula (I), Compound 1, produces dose-dependent (A)antinociception in non-diabetic rats (0.8-20 fmol/Kg, n=3-10 per dose),and (B) to (D): anti-allodynia/antinociception at 10 (B; n=6-8 perdose), 14 (C; n=7-8 per dose) and 24 (D; n=6-7 per dose) weeks post STZadministration in diabetic rats with an approximately 10,000-folddecrease in potency occurring between 10- and 14-wks post-STZadministration. By contrast, vehicle (n=5-7) did not produce significantantinociception or anti-allodynia in the hindpaws of either non-diabeticor STZ-diabetic rats respectively.

FIG. 4 is a graphical representation showing that neither single s.c.bolus doses of vehicle (DMSO:water, 90:10) nor the furoxan NO donor offormula (I), Compound 1, at 80 fmol/kg-8 μmol/kg, significantly alteredmean (±SEM) systolic blood pressure of adult male normotensivenon-diabetic Wistar rats for up to 1 h post-dosing. By contrast, singles.c. bolus doses of Compound 1 at 80 μmol/kg to similar animals,significantly reduced systolic blood pressure at 30 min post-dosingrelative to pre-dosing baseline measurements of systolic blood pressure.

FIG. 5 is graphical representations showing mean (±SEM) ΔPWT versus timecurves in 14-18 week STZ-diabetic rats produced by single s.c. bolusdoses of (A) Compound 1 (800 pmol/Kg) (black upward closed triangles,n=6), naloxone (1.25 μmol/Kg) at 10 minutes prior to Compound 1 (800pmol/Kg) (grey downward closed triangles, n=6), naloxone (1.25 μmol/Kg)(black closed squares, n=2) and vehicle (grey closed circles, n=6) or(B) morphine (7 μmol/Kg) (black upward closed triangles, n=6), naloxone(1.25 μmol/Kg) at 10 minutes prior to morphine (7 μmol/Kg) (greydownward closed triangles, n=7), naloxone (1.25 μmol/Kg) (black closedsquares, n=2) and vehicle (grey closed circles, n=2) showing that in14-18 week STZ-diabetic rats pretreated with naloxone (1.25 μmol/Kgs.c.) at 10 minutes prior to either Compound 1 (800 pmol/Kg s.c.) ormorphine (7 μmol/Kg s.c.) the anti-allodynic effects of morphine wereattenuated by naloxone (as expected) whereas the anti-allodynic effectsof Compound 1 were naloxone insensitive. (C) By contrast theanti-allodynic effects of single s.c. bolus doses of Compound 1 (800pmol/Kg) in 14-18 wk STZ-diabetic rats were partially sensitive topre-treatment with single s.c. bolus doses of ODQ (53 μmol/Kg)administered at 60 minutes prior to administration of Compound 1 [ODQand Compound 1, grey solid squares; vehicle and Compound 1, black solidcircles; vehicle and ODQ, black solid triangles].

FIG. 6 provides graphical representations showing the effects ofCompound 1 on forskolin-stimulated cAMP responses in (A) delta opioidreceptor (DOP)-transfected HEK293 cells (Data are means±SEM, n=4), (B)kappa opioid receptor (KOP)-transfected HEK293 cells (Data aremeans±SEM, n=4) and (C) untransfected HEK293 cells (Data are means±SEM,n=6).

FIG. 7 provides a graphical representation showing the effects ofCompound 1 on forskolin-stimulated cAMP responses in mu-opioid receptor(MOP)-transfected HEK293 cells (Data are means±SEM, n=8).

FIG. 8 provides a graphical representation showing the effects ofpertussis toxin, naloxone or ODQ pretreatment on theforskolin-stimulated cAMP responses evoked by Compound 1 inMOP-transfected HEK293 cells (Data are means±SEM, n=4). ** p<0.01 vsCompound 1 or morphine alone as determined by one-way ANOVA followed byTukey's test.

FIG. 9 is a graphical representation showing that morphine at pM-μMconcentrations inhibited forskolin-stimulated cAMP formation inMOP-transfected HEK-293 cells whereas even lower concentrations ofmorphine produced stimulating responses. Data are presented as mean±SEM(n=3).

FIG. 10 provides a graphical representation showing the displacement ofspecific binding of [³H]-DAMGO by unlabelled DAMGO and Compound 1 inmembranes from MOP-transfected HEK293 cells (Data are means±SEM, n=8).

FIG. 11 is a graphical representation showing that the lack of effectsof 8-Br-cGMP on forskolin-stimulated cAMP responses in MOP-transfectedHEK293 cells.

FIG. 12 is a graphical representation showing that the inhibitoryeffects of Compound 1 on forskolin-stimulated cAMP formation inMOP-transfected HEK293 cells are sensitive to removal of lipidrafts/caveolae from cell membranes.

FIG. 13 is a graphical representation showing mean (±SEM) ΔPWT versustime curves following administration of single s.c. bolus doses of (A)Compound 1 (8 fmol/Kg), (B) morphine (2.8 μmol/Kg) and (C) vehicle, togroups of naïve (black circles) and “tolerant” (grey circles)non-diabetic rats.

FIG. 14 is a graphical representation showing mean (±SEM) ΔPWT vs timecurves following administration of single s.c. bolus doses of (A)Compound 1 (800 pmol/Kg), (B) morphine (2.8 μmol/Kg) and (C) vehicle, togroups of naive (black circles) and “tolerant” (grey circles)STZ-diabetic rats.

FIG. 15 is a graphical representation showing the effects of Compound 33on forskolin-stimulated cAMP responses in mu-opioid receptor(MOP)-transfected HEK293 cells (Data are means±SEM, n=3).

FIG. 16 is a graphical representation showing mean PWT vs time curvesfollowing administration of single s.c. bolus doses of (A) Morphine(2661 nmol/kg) and (B) compound 33 (80, 120, 800 and 1200 nmol/Kg).

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

As used herein, the term “alkyl” refers to a straight chain or branchedsaturated hydrocarbon group having 1 to 10 carbon atoms. Whereappropriate, the alkyl group may have a specified number of carbonatoms, for example, C₁₋₆alkyl which includes alkyl groups having 1, 2,3, 4, 5 or 6 carbon atoms in a linear or branched arrangement. Examplesof suitable alkyl groups include, but are not limited to, methyl, ethyl,n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, 2-methylbutyl,3-methylbutyl, 4-methylbutyl, n-hexyl, 2-methylpentyl, 3-methylpentyl,4-methylpentyl, 5-methylpentyl, 2-ethylbutyl, 3-ethylbutyl, heptyl,octyl, nonyl, and decyl.

The term “alkenyl” as used herein refers to a straight chain or branchedunsaturated hydrocarbon group having 2 to 10 carbon atoms and at leastone double bond. Where appropriate, the alkenyl group may have aspecified number of carbon atoms, for example, C₂₋₆ alkenyl whichinclude alkenyl groups having 2, 3, 4, 5, or 6 carbon atoms in a linearor branched arrangement. Examples of suitable alkenyl groups include,but are not limited to, ethenyl, propenyl, 1-butenyl, 2-butenyl1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl,1,3-pentadienyl, 1,4-pentadienyl, 2,4-pentadienyl, 1-hexenyl, 2-hexenyl,3-hexenyl, 4-hexenyl, 5-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl,1,5-hexadienyl, 2,4-hexadienyl, 1,3,5-hexatrienyl, heptenyl, octenyl,nonenyl and decenyl.

The term “alkynyl” as used herein refers to a straight chain or branchedunsaturated hydrocarbon group having 2 to 10 carbon atoms and at leastone triple bond. Where appropriate, the alkynyl group may have aspecified number of carbon atoms, for example, C₂₋₆ alkynyl whichincludes alkynyl groups having 2, 3, 4, 5 or 6 carbon atoms in a linearor branched arrangement. Examples of suitable alkynyl groups include,but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl,heptynyl, octynyl, nonynyl and decynyl.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the term “about” refers to a quantity, level,concentration, value, dimension, size, or amount that varies by as muchas 30%, 20%, or 10% or even as much as 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or1% to a reference quantity, level, concentration, value, dimension,size, or amount.

The term “allodynia” as used herein refers to pain that results from anon-noxious stimulus i.e., a stimulus that does not normally provokepain. Examples of allodynia include, but are not limited to, coldallodynia, tactile allodynia (pain due to light pressure or touch), andthe like.

The term “analgesia” is used herein to describe states of reduced painperception, including absence from pain sensations as well as states ofreduced or absent sensitivity to noxious stimuli. Such states of reducedor absent pain perception are induced by the administration of apain-controlling agent or agents and occur without loss ofconsciousness, as is commonly understood in the art. The term analgesiaencompasses the term “antinociception”, which is used in the art as aquantitative measure of analgesia or reduced pain sensitivity in animalmodels.

As used herein, the term “aryl” is intended to mean any stable,monocyclic or bicyclic carbon ring of up to 7 atoms in each ring,wherein at least one ring is aromatic. Examples of such aryl groupsinclude, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl,indanyl, biphenyl and binaphthyl.

The term “causalgia” as used herein refers to the burning pain,allodynia and hyperpathia after a traumatic nerve lesion, often combinedwith vasomotor and sudomotor dysfunction and later trophic changes.

By “complex regional pain syndromes” is meant the pain that includes,but is not limited to, reflex sympathetic dystrophy, causalgia,sympathetically maintained pain, and the like.

Throughout this specification, unless the context requires otherwise,the words “comprise,” “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. Thus, use of the term “comprising” and the likeindicates that the listed elements are required or mandatory, but thatother elements are optional and may or may not be present. By“consisting of” is meant including, and limited to, whatever follows thephrase “consisting of”. Thus, the phrase “consisting of” indicates thatthe listed elements are required or mandatory, and that no otherelements may be present. The phrases “consisting essentially of,”“consists essentially of” and the like refer to the components which areessential in order to obtain the advantages of the present invention andany other components present would not significantly change theproperties related to the inventive concept. Put another way, thesephrases refer to the inclusion of any elements listed after the phrase,and limited to other elements that do not interfere with or contributeto the activity or action specified in the disclosure for the listedelements. Thus, the phrases “consisting essentially of,” “consistsessentially of” and the like indicate that the listed elements arerequired or mandatory, but that other elements are optional and may ormay not be present depending upon whether or not they affect theactivity or action of the listed elements.

The term “cycloalkyl” as used herein refers to a cyclic or cagedsaturated hydrocarbon ring having 3 to 8 carbon atoms. Examples ofsuitable cycloalkyl groups include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, norbornyland adamantyl.

The term “cycloalkenyl” as used herein refers to a cyclic or cagedunsaturated hydrocarbon ring having 3 to 8 carbon atoms and at least onedouble bond, but it is not aromatic. Examples of suitable cycloalkenylgroups include, but are not limited to, cyclopropentyl, cyclobutenyl,cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl andcyclooctenyl.

By “effective amount”, in the context of treating or preventing acondition (e.g. a neuropathic condition) is meant the administration ofthat amount of active to an individual in need of such treatment orprophylaxis, either in a single dose or as part of a series, that iseffective for the prevention of incurring a symptom, holding in checksuch symptoms, and/or treating existing symptoms, of that condition. Theeffective amount will vary depending upon the health and physicalcondition of the individual to be treated, the taxonomic group ofindividual to be treated, the formulation of the composition, theassessment of the medical situation, and other relevant factors. It isexpected that the amount will fall in a relatively broad range that canbe determined through routine trials.

As used herein, the term “furoxan” denotes a 1,2,5-oxadiazole-2-oxidecompound with a core structure:

As used herein, the term “halogen” or “halo” refers to fluorine(fluoro), chlorine (chloro), bromine (bromo) and iodine (iodo).

The term “haloalkyl” as used herein refers to an alkyl group as definedabove bearing one or more halo groups. Examples of haloalkyl groupsinclude, but are not limited to, fluoromethyl, chloromethyl,bromomethyl, iodomethyl, difluoromethyl, dichloromethyl,fluorochloromethyl, trifluoromethyl, trichloromethyl, trifluoroethyl,trichloroethyl, pentafluoroethyl and pentachloroethyl.

The term “heterocyclic” or “heterocyclyl” as used herein, refers to acyclic hydrocarbon in which one to four carbon atoms have been replacedby heteroatoms independently selected from N, S, O and Se. Aheterocyclic ring may be saturated or unsaturated and/or may be fused toa carbocyclic, heterocyclic, aryl or heteroaryl ring. Examples ofsuitable heterocyclyl groups include, but are not limited to,tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, pyrrolinyl,pyranyl, piperidinyl, piperazinyl, pyrazolinyl, dithiolyl, oxathiolyl,dioxanyl, dioxinyl, morpholino, thiomorpholino, oxazinyl, azepinyl,diazepinyl, thiazepinyl, oxepinyl and thiapinyl and N-oxides thereof.

The term “heteroaryl” as used herein, represents a stable monocyclic orbicyclic ring of up to 7 atoms in each ring, wherein at least one ringis aromatic and at least one ring contains from 1 to 4 heteroatomsselected from the group consisting of O, N and S. Heteroaryl groupswithin the scope of this definition include; but are not limited to,acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrazolyl, indolyl,benzotriazolyl, furanyl, thienyl, thiophenyl, benzothienyl,benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl,imidazolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl,tetrahydroquinoline, thiazolyl, isothiazolyl, 1,2,4-triazolyl,1,2,4-oxadiazolyl, 1,2,4-thiadiazolyl, benzodioxanyl, benzazepinyl,benzoxepinyl, benzodiazepinyl, benzothiazepinyl and benzothiepinyl.Preferred heteroaryl groups have 5- or 6-membered rings, such aspyrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, imidazolyl,pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, thiazolyl,isothiazolyl, 1,2,4-triazolyl and 1,2,4-oxadiazolyl and1,2,4-thiadiazolyl and N-oxides thereof.

By “hyperalgesia” is meant an increased response to a stimulus that isnormally painful.

As used herein, the term “low-release nitric oxide donor” or“low-release NO donor” is meant any substance that is converted ordegraded or metabolized into, or provides an in vitro source of nitricoxide or NO to deliver a low concentration of nitric oxide into theblood stream. A low-release NO donor may provide rapid or immediaterelease of low levels of NO donor or may provide an initial rapidrelease of low levels of NO donor followed by an extended or graduatedperiod of release of a low concentration of NO. The low-release NO donormay also be a slow-release NO donor. Suitably, the low-release NO donoris administered in an amount such that the NO is delivered into theblood stream in an amount of 10⁻² to 10⁻¹⁵ of a reference concentrationrequired to induce vasodilation in an anatomical site of a referencesubject lacking vascular condition. A suitable amount of low-release NOdonor delivered as a bolus amount is in the range of 0.000001 nmol/Kg to2 nmol/Kg.

By “neuropathic pain” is meant any pain syndrome initiated or caused bya primary lesion or dysfunction in the peripheral or central nervoussystem. Examples of neuropathic pain include, but are not limited to,thermal or mechanical hyperalgesia, thermal or mechanical allodynia,painful diabetic neuropathy, post-herpetic neuralgia, phantom limb pain,sciatica, chemotherapy-induced neuropathy, HIV-AIDS-associatedneuropathy, nerve entrapment pain, and the like.

By “nitric oxide donor,” “NO donor” and the like is meant any substancethat is converted into, degraded or metabolized into, or provides asource of in vivo nitric oxide or NO and includes any and all forms ofNO which exist under physiological conditions. The, the term “NO donor”includes and encompasses any compound which mimics the effects of NO,generates or releases NO through biotransformation, any compound whichgenerates NO spontaneously, any compound which spontaneously releasesNO, or any compound which in any other manner generates NO or a NO-likemoiety when administered to a subject.

“Nociceptive pain” refers to the normal, acute pain sensation evoked byactivation of nociceptors located in non-damaged skin, viscera and otherorgans in the absence of sensitization.

The term “pain” as used herein is given its broadest sense and includesan unpleasant sensory and emotional experience associated with actual orpotential tissue damage, or described in terms of such damage andincludes the more or less localized sensation of discomfort, distress,or agony, resulting from the stimulation of specialized nerve endings.There are many types of pain, including, but not limited to, lightningpains, phantom pains, shooting pains, acute pain, inflammatory pain,neuropathic pain, complex regional pain, neuralgia, neuropathy, and thelike (Dorland's Illustrated Medical Dictionary, 28^(th) Edition, W. B.Saunders Company, Philadelphia, Pa.). The goal of treatment of pain isto reduce the severity of pain perceived by a treatment subject.

By “pharmaceutically acceptable carrier” is meant a solid or liquidfiller, diluent or encapsulating substance that may be safely used intopical, local or systemic administration.

The term “pharmaceutically compatible salt” as used herein refers to asalt which is toxicologically safe for human and animal administration.This salt may be selected from a group including hydrochlorides,hydrobromides, hydroiodides, sulphates, bisulphates, nitrates, citrates,tartrates, bitartrates, phosphates, malates, maleates, napsylates,fumarates, succinates, acetates, terephthalates, pamoates andpectinates.

Base salts include, but are not limited to, those formed withpharmaceutically acceptable cations, such as sodium, potassium, lithium,calcium, magnesium, ammonium and alkylammonium.

Basic nitrogen-containing groups may be quarternised with such agents aslower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides,bromides and iodides; dialkyl sulfates like dimethyl and diethylsulfate; and others.

The term “prodrug” is used in its broadest sense and encompasses thosecompounds that are converted in vivo to a NO donor according to theinvention. Such compounds would readily occur to those of skill in theart, and include, for example, compounds where a free hydroxy group isconverted into an ester derivative. Prodrug forms of compounds may beutilized, for example, to improve bioavailability, mask unpleasantcharacteristics such as bitter taste, alter solubility for intravenoususe, or to provide site-specific delivery of the compound.

By “slow-release nitric oxide donor” or “slow-release NO donor” is meantany substance that is converted or degraded or metabolized into, orprovides a source of in vivo nitric oxide or NO over an extended periodof time, thereby delivering a low concentration of nitric oxide into theblood stream. Suitably the slow-release nitric oxide donor isadministered in an amount such that nitric oxide is delivered at a rateof 0.000001 nmol/kg/hour to 2.0 nmol/kg/hour.

The terms “subject” or “individual” or “patient”, used interchangeablyherein, refer to any subject, particularly a vertebrate subject, andeven more particularly a mammalian subject, for whom therapy orprophylaxis is desired. Suitable vertebrate animals that fall within thescope of the invention include, but are not restricted to, primates,avians, livestock animals (e.g., sheep, cows, horses, donkeys, pigs),laboratory test animals (e.g., rabbits, mice, rats, guinea pigs,hamsters), companion animals (e.g., cats, dogs) and captive wild animals(e.g., foxes, deer, dingoes). A preferred subject is a human in need oftreatment or prophylaxis for a peripheral neuropathic condition,especially PDN. However, it will be understood that the aforementionedterms do not imply that symptoms are present.

The term “sub-normovasodilatory concentration” or “SNC concentration” asused herein refers to a level of NO donor, which enhances NO and thatdoes not alter normal systemic vascular tone in the subject.

The term “sustained-release formulation of nitric oxide donor” as usedherein refers to a formulation of an NO donor that is adapted to releasenitric oxide at a rate of 0.000001 nmol/kg/hour to 2.0 nmol/kg/hour or arange selected from 0.00001 nmol/kg/hour to 2.0 nmol/kg/hour, 0.0002nmol/kg/hour to 1.0 nmol/kg/hour, 0.0005 nmol/kg/hour to 1.0nmol/kg/hour, 0.0001 nmol/kg/hour to 0.5 nmol/kg/hour, 0.0002nmol/kg/hour to 0.2 nmol/kg/hour, 0.0005 nmol/kg/hour to 0.1nmol/kg/hour or 0.001 nmol/kg/hour to 0.05 nmol/kg/hour, 0.005nmol/kg/hour to 0.01 nmol/kg/hour. The sustained release formulation maybe any formulation capable of releasing NO at this rate. Illustrativesustained release formulations are transdermal patches adapted todeliver 0.1 nmol to 500 nmol per 24 hours, especially 10 nmol to 100nmol per 24 hours, more especially 20 nmol to 60 nmol per 24 hours, mostespecially about 50 nmol over 6, 9, 12, 18, 24 or 30 hours.

By “does not alter normal systemic vascular tone” is meant not affectingmean arterial pressure so as to produce inappropriate systemicvasodilation with effects such as hypotension, headache, flushing in anormal subject or in a subject lacking a vascular condition (e.g., aneuropathic condition such as PDN).

Each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl,heterocyclyl and heteroaryl whether an individual entity or as part of alarger entity may be optionally substituted with one or more optionalsubstituents selected from C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl,C₃₋₆cycloalkyl, oxo (═O), C₁₋₆alkyloxy(CH₂)_(p)—,C₂₋₆alkenyloxy(CH₂)_(p)—, C₂₋₆alkynyloxy(CH₂)_(p)—,C₃₋₆cycloalkoxy(CH₂)_(p)—, C₁₋₆alkylthio(CH₂)_(p)—,C₂₋₆alkenylthio(CH₂)_(p)—, C₂₋₆alkynylthio(CH₂)_(p)—,C₃₋₆cycloalkylthio(CH₂)_(p)—, hydroxy(CH₂)_(p)—, —(CH₂)_(p)SH,—(CH₂)_(p)CO₂H, —(CH₂)_(p)CO₂C₁₋₆alkyl, —(CH₂)_(p)CON(R₉)₂,C₂₋₆acyl(CH₂)_(p)—, C₂₋₆acyloxy(CH₂)_(p)—, C₂₋₆alkylSO₂(CH₂)_(p)—,C₂₋₆alkenylSO₂(CH₂)_(p)—, C₂₋₆alkynylSO₂(CH₂)_(p)—, arylSO₂(CH₂)_(p)—,heteroarylSO₂(CH₂)_(p)—, heterocyclylSO₂(CH₂)_(p)—, —(CH₂)_(p)NH₂,—(CH₂)_(p)NH(C₁₋₆alkyl), —(CH₂)_(p)N(C₁₋₆alkyl)₂, —(CH₂)_(p)NH(phenyl),—(CH₂)_(p)N(phenyl)₂, —(CH₂)_(p)NH(acyl), —(CH₂)_(p)N(acyl)(phenyl),—(CH₂)_(p)NH—(CH₂)_(p)—S-aryl, —(CH₂)_(p)N═NHC(O)NH₂, —(CH₂)_(p)C(R₁₀)₃,—(CH₂)_(p)OC(R₁₀)₃, —(CH₂)_(p)SC(R₁₀)₃, —(CH₂)_(p)CN, —(CH₂)_(p)NO₂,—(CH₂)_(p)halogen, —(CH₂)_(p)heterocyclyl, heterocyclyloxy(CH₂)_(p)—,—(CH₂)_(p)heteroaryl, heteroaryloxy(CH₂)_(p)—, —(CH₂)_(p)aryl,—(CH₂)_(p)C(O)aryl and aryloxy(CH₂)_(p)— wherein each R₁₀ isindependently selected from hydrogen and halogen; each R₉ isindependently selected from H, C₁₋₆alkyl, phenyl, cycloalkyl or the twoR₉ taken together with the nitrogen to which they are attached can forma heterocyclyl or heteroaryl ring; and p is 0 or an integer from 1 to 6.Examples of suitable substituents include, but are not limited to,methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, vinyl,oxo (═O), methoxy, ethoxy, propoxy, isopropoxy, butoxy, methylthio,ethylthio, propylthio, isopropylthio, butylthio, hydroxy, hydroxymethyl,hydroxyethyl, hydroxypropyl, hydroxybutyl, fluoro, chloro, bromo, iodo,cyano, nitro, —CO₂H, —CO₂CH₃, —CO₂CH₂CH₃—CH₂CO₂CH₃, trifluoromethyl,trifluoromethoxy, trifluoromethylthio, acetyl, morpholino, amino,methylamino, dimethylamino, phenyl, phenylcarbonyl, NHCOphenyl,NHCObenzyl.

2. Methods for the Production of Analgesia and Compounds of theInvention

The present invention provides methods for producing analgesia in asubject having a neuropathic condition. These methods generally compriseadministering to the subject at least one NO donor at a level thatenhances NO and that does not alter normal systemic vascular tone in thesubject. Suitably, this level equates to one that does not inducevasodilation, or not appreciably, in “healthy” or non-NO deficientcirculations. Suitably, the level of NO is a sub-normovasodilatory (SNV)concentration that ranges from about ½ to about 10⁻¹⁵ of those currentlyused in clinical applications.

The method of the present invention has particular utility in theprevention and/or alleviation of the painful symptoms associated withneuropathic conditions. There are many possible causes of neuropathicconditions and it will be understood that the present inventioncontemplates the treatment and/or prevention of pain associated with anyneuropathic condition regardless of the cause. In one embodiment, theneuropathic conditions are a result of diseases of the nerves (primaryneuropathy) and neuropathy that is caused by systemic disease (secondaryneuropathy), such as but not limited to diabetic neuropathy, HerpesZoster (shingles)-related neuropathy, phantom limb pain,uraemia-associated neuropathy, amyloidosis neuropathy, HIV sensoryneuropathies, hereditary motor and sensory neuropathies (HMSN),hereditary sensory neuropathies (HSNs), hereditary sensory and autonomicneuropathies, hereditary neuropathies with ulcero-mutilation,nitrofurantoin neuropathy, tomaculous neuropathy, neuropathy caused bynutritional deficiency and neuropathy caused by kidney failure. Othercauses include repetitive activities such as typing or working on anassembly line, medications known to cause peripheral neuropathy such asseveral AIDS drugs (DDC and DDI), antibiotics (metronidazole, anantibiotic used for Crohn's disease, isoniazid used for tuberculosis),gold compounds (used for rheumatoid arthritis), some chemotherapy drugs(such as cisplatin, vincristine and others) and many others. Chemicalcompounds are also known to cause peripheral neuropathy includingalcohol, lead, arsenic, mercury and organophosphate pesticides. Someperipheral neuropathies are associated with infectious processes (suchas Guillian-Barre syndrome). In another embodiment, the neuropathiccondition is a peripheral neuropathic condition such as PDN or relatedcondition.

The neuropathic condition may be acute or chronic and, in thisconnection, it will be understood by persons of skill in the art thatthe time course of a neuropathy will vary, based on its underlyingcause. With trauma, the onset of symptoms may be acute, or sudden, withthe most severe symptoms being present at the onset or developingsubsequently. Inflammatory and some metabolic neuropathies have asubacute course extending over days to weeks. A chronic course overweeks to months usually indicates a toxic or metabolic neuropathy. Achronic, slowly progressive neuropathy over many years occurs with mosthereditary neuropathies or with a condition termed chronic inflammatorydemyelinating polyradiculoneuropathy (CIDP). Neuropathic conditions withsymptoms that relapse and remit include the Guillian-Barre syndrome.

The NO donor includes and encompasses any substance that is convertedinto, or degraded or metabolized into, or provides a source of, in vivoNO, inclusive of NO in its various redox forms. The presence of nitricoxide (NO) in biological systems is usually inferred based on itsphysiological effect. However, several different redox forms of NO suchas the NO free radical (NO⁻), the nitrosonium cation (NO⁺), the nitroxylanion (NO⁻) or other oxides of nitrogen (NOx) are known to exist underphysiological conditions and there is no clear evidence to suggest thatone form is favored over another (Butler et al. 1995, Trends Pharmacol.Sci. 16:18-22; Stamler et al. 1992, Science 258:1898-1902). NO is alsoknow to react with thiols to form S-nitrosothiols (RS—NO) and mayrepresent a long-term storage form for NO. The category of NO donorsincludes compounds having differing structural features. For example, NOdonors include, but are not limited to: metabolic precursors of NO suchas L-arginine and L-citrulline; so-called “organonitrates” such asnitroglycerin (GTN), glyceryl trinitrate, isosorbide 5-mononitrate(ISMN), isosorbide dinitrate (ISDN), pentaerythritol tetranitrate(PETN), erythrityl tetranitrate (ETN), amino acid derivatives such asN-hydroxy-L-arginine (NOHA), N⁶-(1-iminoethyl)lysine) (L-NIL),L-N⁵-(1-iminoethyl)omithine (LN-NIO), N-methyl-L-arginine (L-NMMA), andS-nitrosoglutathione (SNOG); other compounds which generate or releaseNO under physiologic conditions such as S,S-dinitrosodithiol (SSDD),[N-[2-(nitroxyethyl)]-3-pyridinecarboxamide (nicorandil), sodiumnitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP),3-morpholino-sydnonimine (SIN-1), molsidomine, DEA-NONOate(2-(N,N-diethylamino)-diazenolate-2-oxide), spermine NONOate(N-[4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl-1,3-propanediamine),and NO gas, or a functional equivalent thereof. The organic nitratesGTN, ISMN, ISDN, ETN, and PETN, as well as nicorandil are commerciallyavailable in pharmaceutical dosage forms. SIN-1, SNAP,S-thioglutathione, L-NMMA, L-NIL, L-NIO, spermine NONOate, andDEA-NONOate are commercially available from Biotium, Inc. 183 ShorelineCourt, Richmond, Calif., USA. In other embodiments, the NO donor issuitably selected from [3-(1H-Imidazol-4-yl)propyl]guanidines-containingfuroxan moieties, as for example, described in Bertinaria et al. (2003,Bioorganic & Medicinal Chemistry 11: 1197-1205), NO-donor phenols asdescribed, for example, in Boschi et al. (2006, J. Med. Chem. 49:2886-2897), pseudojujubogenin glycosides such as dammarane-typetriterpenoid saponins (e.g., bacopasaponins) as well as theirderivatives or analogues.

In one embodiment of the present invention, the at least one NO donor isa furoxan NO donor, particularly a furoxan compound of formula (I):

wherein R₁ and R₂ are independently selected from hydrogen, alkyl,alkenyl, alkynyl, haloalkyl, —C₀₋₆alkylcycloalkyl,—C₀₋₆alkylcycloalkenyl, —C₀₋₆alkylaryl, —C₀₋₆alkylheterocyclyl,—C₀₋₆alkylheteroaryl, —C₀₋₆alkylCO₂R₃, —C₀₋₆alkylC(O)R₃,—C₀₋₆alkylC(O)NHR₄, —C₀₋₆alkylN(R₄)₂, —C₀₋₆alkylN⁺(R₇)₃, —C₀₋₆alkylOR₅,—C₀₋₆alkylSR₅, —C₀₋₆alkylC(═NR₆)R₃, —C₀₋₆alkylN═NR₅,—C₀₋₆alkylNR₄N(R₄,)₂, —C₀₋₆alkylNR₄C(═NR₄)N(R₄,)₂, —C₀₋₆alkylhalo,—C₀₋₆alkylS(O)R₃, —C₀₋₆alkylSO₂R₃, —CN and —NO₂; or R₁ and R₂ takentogether form an optionally substituted 5 to 8 membered saturated orunsaturated carbocyclic or heterocyclic ring, an aryl ring or aheteroaryl ring;R₃ is selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl,—C₀₋₆alkylcycloalkyl, —C₀₋₆alkylcycloalkenyl, —C₀₋₆alkylaryl,—C₀₋₆alkylheterocyclyl, —C₀₋₆alkylheteroaryl, —C₁₋₆alkylCO₂R₇,—C₀₋₆alkylN(R₄)₂, —C₁₋₆alkylNR₄C(═NR₄)N(R₄,)₂ —C₁₋₆alkylOR₇ and—C₁₋₆alkylSR₇;each R₄ is selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl,—C₀₋₆alkylcycloalkyl, —C₀₋₆alkylcycloalkenyl, —C₀₋₆alkylaryl,—C₀₋₆alkylheterocyclyl, —C₀₋₆alkylheteroaryl, —C₀₋₆alkylC(O)R₈,—C₀₋₆alkylC(S)R₈, —C₀₋₆alkylCO₂R₇, —C₀₋₆alkylSO₂R₈ and —C₀₋₆alkylOR₇;R₅ is selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl,—C₀₋₆alkylcycloalkyl, —C₀₋₆alkylcycloalkenyl, —C₀₋₆alkylaryl,—C₀₋₆alkylheterocyclyl, —C₀₋₆alkylheteroaryl, —C₀₋₆alkylC(O)R₇,—C₀₋₆alkylCO₂R₈, —C₀₋₆alkylN(R₇)₂, —C₀₋₆alkylC(O)N(R₇)₂,—C₀₋₆alkylNR₄C(═NR₄)N(R₄,)₂, —C₁₋₆alkylOR₇ and —C₁₋₆alkylSR₇;R₆ is selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl,—C₀₋₆alkylcycloalkyl, —C₀₋₆alkylcycloalkenyl, —C₀₋₆alkylaryl,—C₀₋₆alkylheterocyclyl, —C₀₋₆alkylheteroaryl, —C₀₋₆alkylNHC(O)N(R₇)₂,—C₀₋₆alkylNHC(O)R₇, —C₀₋₆alkylNHSO₂R₇, —C₀₋₆alkylNHCO₂R₇,—C₀₋₆alkylOC(O)R₇, —C₀₋₆alkylC(O)R₇, —CN and —OR₇;each R₇ is selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl,—C₀₋₆alkylcycloalkyl, —C₀₋₆alkylcycloalkenyl, —C₀₋₆alkylaryl,—C₀₋₆alkylheterocyclyl and —C₀₋₆alkylheteroaryl; andR₈ is selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl,—C₀₋₆alkylcycloalkyl, —C₀₋₆alkylcycloalkenyl, —C₀₋₆alkylaryl,—C₀₋₆alkylheterocyclyl, —C₀₋₆alkylheteroaryl and —N(R₇)₂;wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl,heterocyclyl and heteroaryl group is optionally substituted; or apharmaceutically compatible salt thereof.

In some embodiments, R₁ and R₂ are independently selected from—C₁₋₆alkyl, —C₃₋₈cycloalkyl, aryl, —COaryl, —C(O)H, —C(O)alkyl,—C(O)haloalkyl, —NH₂, —NO₂, —Salkyl, —Saryl, —SO₂alkyl, —SO₂aryl, —CO₂H,—CO₂alkyl, —CO₂haloalkyl, —CO₂aryl, —NHC(O)alkyl, —NHC(O)aryl, —N═NH,—N═Nalkyl, —N═Naryl, —C(═NOH)H, —OH, —Oalkyl, —Oaryl, —OC(O)alkyl,—OC(O)aryl, —OC(O)NH₂, —OC(O)NH(alkyl), —OC(O)N(alkyl)₂,—OC(O)N(alkyl)(aryl), —OC(O)N(aryl)₂, —OC(O)NH(aryl), —CH═NNHCO₂alkyl,—CH═NNHCO₂aryl, —CH═NNHC(O)NH₂, —CH═NNHC(O)NH(alkyl),—CH═NNHC(O)N(alkyl)₂, —CH═NNHC(O)NH(aryl), —CH═NNHC(O)N(alkyl)(aryl),—CH═NNHC(O)N(aryl)₂, —CH═NNHSO₂alkyl, —CH═NNHSO₂aryl, —CN, —CH₂OH,—C(O)NH₂, —C(O)NH(alkyl), —C(O)N(alkyl)₂, —C(O)NH(aryl),—C(O)N(alkyl)(aryl), —C(O)N(aryl)₂, —OalkylNH₂, —OalkylNH(alkyl),—OalkylN(alkyl)₂, —OalkylNHaryl, —OalkylN(aryl)₂, —OalkylN(aryl)(alkyl),—SalkylNH₂, —SalkylNH(alkyl), —SalkylN(alkyl)₂, —SalkylNHaryl,—SalkylN(aryl)₂, —SalkylN(aryl)(alkyl), —CH₂NH₂, —CH₂NH(alkyl),—CH₂N(alkyl)₂, —CH₂NH(aryl), —CH₂N(alkyl)(aryl), —CH₂N(aryl)₂,—C(O)heterocyclyl, —C(O)heteroaryl, —C(O)hetercyclyl-heteroaryl,—C(O)heteroaryl-heterocyclyl, —C(O)heterocyclyl-heterocyclyl,—C(O)heteroaryl-heteroaryl, —OalkylNHalkylOaryl, —OalkylNHC(O)heteroaryland —OalkylOC(O)aryl, wherein each alkyl, aryl, heterocyclyl andheteroaryl is optionally substituted, or where R₁ and R₂ taken togetherwith the carbon atoms to which they are attached form an optionallysubstituted 5 or 6 membered carbocyclic, aryl, heterocyclic orheteroaryl ring.

In some embodiments, R₁ and R₂ are independently selected from methyl,ethyl, propyl, isopropyl, cyclopentyl, cyclohexyl, phenyl,4-fluorophenyl, 4-methoxyphenyl, 4-CH₃SO₂phenyl-, 4-NH₂SO₂phenyl-,4-CH₃Sphenyl, phenylSO₂-, phenylS—, —NO₂, —NH₂, —C(O)H, —C(O)CH₃,—C(O)CF₃, —C(O)phenyl, —C(O)-4-fluorophenyl, —C(O)-4-chlorophenyl,—C(O)-2-chlorophenyl, —C(O)-2-methylphenyl, —C(O)-4-methylphenyl,—C(O)-2-methoxyphenyl, —C(O)-4-methoxyphenyl, —C(O)-4-cyanophenyl,—C(O)-3-cyanophenyl, —CO₂H, —NHC(O)CH₃, —N═N—H, —C(═NOH)H, —OH,—CH═NNHC(O)NHbutyl, —CH═NNHSO₂phenyl, —CH═NNHC(O)NHaryl, —CN,—OC(O)N(CH₃)(propyl), —CH₂OH, —C(O)NH₂, —OCH₂CH₂N(CH₃)₂, —SCH₂CH₂NH₂,—SCH₂CH₂N(CH₃)₂, —Omethyl, Oethyl, —CH₂N(CH₃)₂,—NHpropylOphenylCH₂—N-piperidine,—C(O)-1-piperidine-4-[2-(4-amino-6,7-dimethoxy-quinazoline)],—OpropylNH-(2-hydroxypropyl)oxynaphthalene, —Opropyl-NHC(O)-3-pyridineand O-alkylOC(O)-(2-acetylphenyl), or R₁ and R₂ taken together with thecarbon atoms to which they are attached form a cyclopentyl ring,2-oxo-cyclopentyl ring, cyclohexyl ring, 2-oxo-cyclohexyl ring, phenylring, 3-fluorophenyl ring, 3-methoxy-phenyl ring, 2N-pyridine ring or3-N-pyridine ring.

In some embodiments, R₁ is not —C(O)H, especially when R₂ is methyl.

In some embodiments, the furoxan compound of formula (I) is4-formyl-3-methyl-1,2,5-oxadiazole-2-oxide (Compound 1).

In some embodiments, R₁ and/or R₂ include a carbonyl or hydroxylsubstituent, such as —C₀₋₆alkylCO₂R₃, —C₀₋₆alkylC(O)R₃,—C₀₋₆alkylC(O)NHR₄, —C₀₋₆alkylC(O)N(R₄)₂, —C₀₋₆alkylOH where R₃ and R₄are as defined in formula (I) or where R₁ and R₂ taken together form a5-8 membered saturated or unsaturated carbocyclic or heterocyclic ringsubstituted with an oxo (═O) group, especially where R₁ or R₂ are —CO₂H,—C(O)H, —C(O)NH₂, —C(O)CH₃, —C(O)CF₃, —CH₂OH, or where R₁ and R₂ takentogether form a 5 or 6 membered carboxyclic ring substituted with an oxo(═O) group.

In some embodiments R₁ and/or R₂ include an optionally substitutedphenyl ring, especially an unsubstituted phenyl ring or a phenyl ringsubstituted with at least one substituent selected from alkyl, hydroxy,alkoxy, halo and cyano, especially alkoxy and halo, more especiallymethoxy and fluoro.

In some embodiments one of R₁ and R₂ include a carbonyl or hydroxylsubstituent and the other includes an optionally substituted phenylring.

In some embodiments, the compounds of formula (I) are selected fromthose in Table 1.

TABLE 1 Compound R₁ R₂  1 —C(O)H —CH₃  2 —CH₂—CH₂—CH₂—CH₂—  3

 4

 5 —Ph —Ph  6 —CO₂H —CH₃  7 —C(O)NH₂ —CH₃  8 —CO₂H —CH₂CH₃  9 —C(O)NH₂—CH₂—CH₃ 10 —CO₂H —CH(CH₃)₂ 11 —C(O)NH₂ —CH(CH₃)₂ 12 —C(O)CH₃ —CH₃ 13—Ph —CH₃ 14 —Ph —C(O)H 15 —Ph-4-OCH₃ —C(O)H 16 —C(O)CH₂CH₂— 17—C(O)CH₂CH₂CH₂— 18 —Ph-4-F —CO₂H 19

20

21 —Ph-4-F —Ph-4-F 22 —C(O)CF₃ —Ph 23 —Ph-4-F —CH₃ 24 —Ph-4-OCH₃ —CH₃ 25—Ph —CO₂H 26 —Ph —C(O)NH₂ 27 —Ph —C(O)CH₃ 28 —Ph-4-F —C(O)H 29 —Ph-4-F—C(O)NH₂ 30 —Ph-4-OCH₃ —C(O)NH₂ 31 —Ph —CH₂OH 32 —Ph-4-OCH₃ —CH₂OH

The compounds listed in Table 1 may be isomeric in that the substituentlisted as R₁ may be substituted at R₂ and the substituent listed as R₂may be substituted at R₁.

In some cases, the furoxans of the invention are able to isomerizebetween the 2-N-oxide and 5-N-oxide compounds as shown in Scheme 1:

(Katritsky & Rees, Comprehensive Heterocyclic Chemistry, 1984, 6,403-404, Pergammon Press; Advances in Heterocyclic Chemistry, 1981,29:289-297).

In one aspect of the invention there is provided compounds of formula(I) as described above.

The furoxan compounds used in the present invention may be synthesizedby methods known in the art (Katritsky & Rees, ComprehensiveHeterocyclic Chemistry, 1984, 6, 420-425, Pergammon Press; Advances inHeterocyclic Chemistry, 1981, 29:270-284). Monocyclic furoxans may beprepared by, for example, oxidation of α-dioximes, dehydration ofα-nitro ketone oximes, dimerization of nitrile oxides, nitration of1,2-dialkylvinyl azides, loss of nitrous acid from nitrolic acids and byreaction of olefins and nitrogen oxides. Benzofuroxans and otheraromatic ring fused furoxans are commonly prepared by pyrolysis ofortho-nitrophenylazides, oxidation of ortho-nitroanilines and oxidationof ortho-quinone dioximes. Cycloalkyl fused furoxans can be prepared byreacting an α-bromo cyclic ketone with hydroxylamine followed byoxidation or by treating a cyclic 1,3-dione with sodium nitrite followedby hydroxylamine, the resulting trioxime can be then be treated withsodium hypobromide to form the fused furoxan.

In some cases, asymmetric isomeric furoxans may be prepared selectivelyby oxidation of geometric isomers of the starting asymmetric dioximes.an example of such a reaction is given in Scheme 2.

This reaction tolerates a variety of substituents as R_(a) and R_(b).For Example, R_(a) and R_(b) may be independently alkyl, aryl, acyl,amino and halogen. Such substituents may be optionally furtherderivatised after formation of the furoxan ring.

For example, an appropriate α,β-unsaturated aldehyde may be reacted withsodium nitrite in mild acid as shown in Scheme 3:

(J. Heterocyclic Chem., 1989, 29(5):1345-47)

The aldehyde may be further elaborated by methods known in the art, forexample, oxidation to a carboxylic acid and optional reaction with analkyl or aryl group to provide an ester or an amino group to provide anamide. Another option is to form an acid chloride followed by furtherreaction with an alkyl or aryl group to form an acyl group or reactionwith an amine to form an amide group. A further option is to react theadehyde group with an amino group to form an imine which may beoptionally reduced. Another option, especially when R is aromatic, is toreact the aldehyde with an allylic alcohol.

Another means of preparing furoxan compounds is nitric acid oxidation ofsubstituted acetophenone compounds as shown in Scheme 4:

(Nitrode et al., Bioorg. Med. Chem. Lett., 2006, 16:2299-2301)

Other syntheses of furoxans are also known. (Cerecetto and Porcal,Mini-reviews in Medicinal Chemistry, 2005, 5:57-71).

In some embodiments, the NO donor is a compound of formula (II):

X is a covalent bond, —O—, —S— or —N(R₂₃)—;W is a covalent bond, —O—, —S—, —N(R₂₃)— or —C₆H₄—;Y is a covalent bond, —O— or —S—;Z is —NO or —NO₂R₂₀ is hydrogen, —OH, —Oalkyl, —NH₂, —NHalkyl or —N(alkyl)₂;each R₂₁ and R₂₂ is independently selected from hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, halo, hydroxyl, alkyloxy, —CO₂H,—CO₂alkyl, —CONH₂, —CONHalkyl, —CON(alkyl)₂, aryl, heterocyclyl andheteroaryl;R₂₃ is a hydrogen or alkyl;n is 0 or an integer of 1-10; andm is an integer of 1-10.

In some embodiments, one or more of the following applies

X is a covalent bond, —O—, —S—, especially a covalent bond or —O—,

W is a covalent bond, —O—, —S— or —C₆H₄—, especially a covalent bond or—O—; more especially a covalent bond

Y is a covalent bond or —O—, especially —O—;

Z is NO₂;

R₂₀ is hydrogen, —OH or NH₂; especially —OH;

each R₂₁ and R₂₂ is hydrogen, alkyl, hydroxyl or alkoxy; especiallyhydrogen;

R₂₃ is hydrogen; and

n+m is an integer from 3-7, especially 4.

In a particular embodiment, the NO donor is a compound of (II) in whichR₂₀ is —OH, X is a covalent bond, W is a covalent bond, Y is —O—, Z isNO₂, each R₂₁ and R₂₂ is hydrogen and m+n is an integer from 3-7,especially 5-nitratopentanoic acid.

In one aspect of the invention there is provided compounds of formula(II) as described above.

These nitrato compounds may be prepared by methods known in the art. Forexample a suitable bromoalkyl carboxylic acid can be reacted with anitrate salt such as silver nitrate under anhydrous conditions, asdescribed in EP 0984012 A2.

In accordance with the present invention, an NO donor is administered ata level that enhances NO and that does not alter normal systemicvascular tone in the subject. Suitably, the level of NO is asub-normovasodilatory (SNV) concentration that ranges from about ½ toabout 10⁻¹⁵ of a reference concentration required to induce vasodilationin an anatomical site of a reference subject lacking a vascularcondition, which is suitably the neuropathic condition. Vasodilation maybe measured using any suitable technique for defining SNVconcentrations. Illustrative methods for measuring vasodilation include,but are not limited to, measuring systolic blood pressure (e.g., in alimb or tail), by measuring blood flow in ears or using the vasodilationassay described in Pharmacol Res. 39(3): 217-20 (1999). In a specificembodiment, systolic blood pressure is measured in normotensiveexperimental animals (e.g., rats) that are lightly sedated viaintraperitoneal injection of Zoletil (tiletamine 15 mg/kg, zolazepam 15mg/kg), using an inflatable tail-cuff. Representative SNV concentrationranges include from about ½ to about 1/20, ½ to about 1/50, ½ to about10⁻¹, 10⁻¹ to about 10⁻¹⁵, 10⁻² to about 10⁻¹⁵, 10⁻³ to about 10⁻¹⁵,10⁻⁴ to about 10⁻¹⁵, 10⁻⁵ to about 10⁻¹⁵, 10⁻⁶ to about 10⁻¹⁵, 10⁻² toabout 10⁻¹³, 10⁻² to about 10⁻¹², 10⁻² to about 10⁻¹¹, 10⁻² to about10⁻¹⁰, 10⁻² to about 10⁻⁹, 10⁻² to about 10⁻⁸, 10⁻² to about 10⁻⁷, or10⁻² to about 10⁻⁶ of the reference concentration. In some embodimentsin which the NO donor is in slow-release form, the amount of NO donorthat is administered as a bolus is in the range of 0.000001 nmol/kg to 2nmol/kg, 0.00001 nmol/kg to 2 nmol/kg, 0.0001 nmol/kg to 2 nmol/kg,0.001 nmol/kg to 2 nmol/kg, 0.001 nmol/kg to 1 nmol/kg, 0.001 nmol/kg to0.6 nmol/kg, 0.004 nmol/kg to 0.4 nmol/kg, preferably in a rangeselected from 0.005 nmol/kg to 0.3 nmol/kg, 0.006 nmol/kg to 0.2nmol/kg, 0.007 nmol/kg to 0.1 nmol/kg, 0.008 nmol/kg to 0.09 nmol/kg,0.009 nmol/kg to 0.08 nmol/kg, 0.01 nmol/kg to 0.07 nmol/kg, 0.02nmol/kg to 0.06 nmol/kg, and especially 0.03 nmol/kg to 0.05 nmol/kg. Inother embodiments in which the NO donor is formulated in a sustainedrelease formulation, the NO donor is adapted to release nitric oxide ata rate of 0.00001 nmol/kg/hour to 2.0 nmol/kg/hour, 0.0001 nmol/kg/hourto 2.0 nmol/kg/hour, 0.0002 nmol/kg/hour to 2.0 nmol/kg/hour or in arange selected from 0.001 nmol/kg/hour to 1.0 nmol/kg/hour, 0.005nmol/kg/hour to 1.0 nmol/kg/hour, 0.001 nmol/kg/hour to 0.5nmol/kg/hour, 0.002 nmol/kg/hour to 0.2 nmol/kg/hour, 0.005 nmol/kg/hourto 0.1 nmol/kg/hour, or 0.01 nmol/kg/hour to 0.05 nmol/kg/hour. Inillustrative examples of this type, the NO donor is a transdermal patchadapted to release 0.5 nmol to 500 nmol, especially 10 nmol to 100 nmol,more especially 20 nmol to 60 nmol and even more especially about 50nmol over 6, 9, 12, 18, 24 or 30 hours.

Advantageously, in some embodiments, particularly embodiments where theNO donor is a furoxan NO donor, the NO donor may be used in a mannerthat minimizes analgesic tolerance development. While tolerance to theanalgesic effects of NO donors may occur upon continuous dosing, uponceasing dosing the analgesic sensitivity to the NO donor returns tonormal allowing further administration at original low levels observedin a naive patient. In a clinically relevant setting, where a subjectwould be dosed on an intermittent dosing schedule, such as once daily,twice daily or three times daily, even over an extended period of time,tolerance to the NO donor is likely to be minimized. This is in contrastto the use of traditional opioid analgesics where tolerance developswith long term administration and the subject requires ever increasingdoses.

In some embodiments, the NO donor is a compound that is a slow releasenitric oxide donor, releasing NO over an extended period of time afteradministration. Such a nitric oxide donor maintains a very low level ofnitric oxide in the blood stream. The slow release nitric oxide donormay deliver NO at a rate of 0.000001 nmol/Kg/hr to 2.0 nmol/Kg/hr.

In some embodiments, the NO donor is a compound that is a low-releasenitric oxide donor, releasing a low concentration of NO into the bloodstream by rapid or immediate release of the NO or by a graduated releasewhere the release rate of NO is not constant or by slow-release of theNO. The low-release NO donor may deliver NO to the blood stream of aconcentration of 10⁻² to 10⁻¹⁵ of a reference concentration required toinduce vasodilation in an anatomical site of a reference subject lackingvascular condition. For example, the low-release NO donor may beadministered as a bolus in the range of 0.000001 nmol/Kg to 2 nmol/Kg.

Suitably, the level of NO donor administered is effective for treatingor preventing a neuropathic condition, including a peripheralneuropathic condition such as PDN or a related condition, and especiallyfor the treatment or prevention of pain in neuropathic conditions,including the prevention of incurring pain, holding pain in check,and/or treating existing pain. Whether pain has been treated isdetermined by measuring one or more diagnostic parameters which isindicative of pain (e.g., subjective pain scores, tail-flick tests andtactile allodynia) compared to a suitable control. In the case of ananimal experiment, a “suitable control” is an animal not treated withthe nitric oxide donor, or treated with the pharmaceutical compositionwithout nitric oxide donor. In the case of a human subject, a “suitablecontrol” may be the individual before treatment, or may be a human(e.g., an age-matched or similar control) treated with a placebo. Inaccordance with the present invention, the treatment of pain includesand encompasses without limitation: (i) preventing pain experienced by asubject which may be predisposed to the condition but has not yet beendiagnosed with the condition and, accordingly, the treatment constitutesprophylactic treatment for the pathologic condition; (ii) inhibitingpain initiation or a painful condition, i.e., arresting its development;(iii) relieving pain, i.e., causing regression of pain initiation or apainful condition; or (iv) relieving symptoms resulting from a diseaseor condition believed to cause pain, e.g., relieving the sensation ofpain without addressing the underlying disease or condition.

In some embodiments, the NO donor may be administered in combinationwith, simultaneously in one composition or in separate compositions, orseparately and sequentially, with another treatment for neuropathicpain, especially when the NO donor is a furoxan NO donor of formula (I).In some embodiments, the other treatment for neuropathic pain is not anopioid analgesic such as morphine. In some embodiments, the othertreatment for neuropathic pain is selected from one or more ofanticonvulsants such as carbamazepine, gabapentin, phenytoin, pregabalinand valproate; antidepressants such as amitriptyline, desipramine andduloxetine; central α-2 adrenergic agonists such as clonidine andtizanidine; corticosteroids such as dexamethasone and prednisone;NMDA-receptor antagonists such as memantin and dextromethorphan, oralsodium channel blockers such as mexiletine or topical compositions suchas capsaicin, EMLA® and lidocaine; or other compositions includingbaclofen and pamidronate.

In some embodiments, the NO donor is administered without the need foradministration of an opioid analgesic. In other embodiments,particularly when the NO donor is a furoxan NO donor of formula (I), theNO donor may be administered in combination with, simultaneously in onecomposition or in separate compositions, or separately and sequentially,with an opioid analgesic, especially a μ-opioid analgesic, such asmorphine.

3. NO Donor-Containing Compositions

Another aspect of the present invention provides compositions forproducing analgesia and especially for treating, preventing and/oralleviating the painful symptoms of a neuropathic condition. Theseanalgesic compositions generally comprise at least one NO donor at alevel that enhances NO and that does not alter normal systemic vasculartone in the subject, as broadly described above. The effect of thecompositions of the present invention may be examined by using one ormore of the published models of pain/nociception or of neuropathy,especially peripheral neuropathy, and more especially PDN, known in theart. This may be demonstrated, for example using an animal model whichassesses the onset and development of tactile allodynia, the definingsymptom of PDN, as for example described herein. The analgesic activityof the compounds of this invention can be evaluated by any method knownin the art. Examples of such methods are the Tail-flick test (D'Amour etal. 1941, J. Pharmacol. Exp. and Ther. 72: 74-79); the Rat TailImmersion Model, the Carrageenan-induced Paw Hyperalgesia Model, theFormalin Behavioral Response Model (Dubuisson et al. 1977, Pain 4:161-174), the Von Frey Filament Test (Kim et al. 1992, Pain 50:355-363), the Chronic Constriction Injury, the Radiant Heat Model, andthe Cold Allodynia Model (Gogas et al. 1997, Analgesia 3: 111-118), thepaw pressure test of mechanical hyperalgesia (Randall and Selitto, 1997,Arch Int Pharmacodyn 111: 409-414; Hargreaves et al. 1998, Pain, 32:77-88). An in vivo assay for measuring the effect of test compounds onthe tactile allodynia response in a rat model of painful diabeticneuropathy is described in Example 1. Compositions which test positivein such assays are particularly useful for the treatment or preventionof neuropathic pain.

The NO donors may be provided as salts with pharmaceutically compatiblecounterions. Pharmaceutically compatible salts may be formed with manyacids, including but not limited to hydrochloric, sulfuric, acetic,lactic, tartaric, maleic, succinic, etc. Salts tend to be more solublein aqueous or other protonic solvents that are the corresponding freebase forms.

The dose of NO donor administered to a patient should be sufficient toachieve a beneficial response in the patient over time such as areduction in, or relief from, pain, especially neuropathic pain. Thequantity of the compound(s) to be administered may depend on the subjectto be treated inclusive of the age, sex, weight and general healthcondition thereof. This quantity, however, will be one that enhances NOand that does not alter normal systemic vascular tone in the subject. Inthis regard, precise amounts of the NO donor(s) for administration willdepend on the judgement of the practitioner. In determining theeffective amount of the NO donor(s) to be administered in the productionof analgesia, the physician may evaluate severity of the pain symptomsassociated with nociceptive or inflammatory pain conditions or numbness,weakness, pain, loss of reflexes and tactile allodynia associated withneuropathic conditions, especially peripheral neuropathic conditionssuch as PDN. In any event, those of skill in the art may readilydetermine suitable dosages of the nitric oxide donors of the inventionwithout undue experimentation.

In some embodiments, and dependent on the intended mode ofadministration, the NO donor-containing compositions will generallycontain about 0.001% to 90%, about 0.1% to 50%, or about 1% to about25%, by weight of NO donor, the remainder being suitable pharmaceuticalcarriers and/or diluents etc. The dosage of the nitric oxide donor candepend on a variety of factors, such as the individual nitric oxidedonor, mode of administration, the species of the affected subject, ageand/or individual condition.

Depending on the specific neuropathic condition being treated, the NOdonor(s) may be formulated and administered systemically, topically orlocally. Techniques for formulation and administration may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition. Suitable routes may, for example, include buccal,sublingual, oral, rectal, transmucosal, or intestinal administration;parenteral delivery, including intramuscular, subcutaneous,intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections. For injection, the therapeutic agents of theinvention may be formulated in aqueous solutions, suitably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiological saline buffer. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart.

Alternatively, the compositions of the invention can be formulated forlocal or topical administration. In this instance, the subjectcompositions may be formulated in any suitable manner, including, butnot limited to, creams, gels, oils, ointments, solutions andsuppositories. Such topical compositions may include a penetrationenhancer such as benzalkonium chloride, digitonin, dihydrocytochalasinB, capric acid, increasing pH from 7.0 to 8.0. Penetration enhancerswhich are directed to enhancing penetration of the active compoundsthrough the epidermis are advantageous in this regard. Alternatively,the topical compositions may include liposomes in which the activecompounds of the invention are encapsulated.

The compositions of this invention may be formulated for administrationin the form of liquids, containing acceptable diluents (such as salineand sterile water), or may be in the form of lotions, creams or gelscontaining acceptable diluents or carriers to impart the desiredtexture, consistency, viscosity and appearance. Acceptable diluents andcarriers are familiar to those skilled in the art and include, but arenot restricted to, ethoxylated and nonethoxylated surfactants, fattyalcohols, fatty acids, hydrocarbon oils (such as palm oil, coconut oil,and mineral oil), cocoa butter waxes, silicon oils, pH balancers,cellulose derivatives, emulsifying agents such as non-ionic organic andinorganic bases, preserving agents, wax esters, steroid alcohols,triglyceride esters, phospholipids such as lecithin and cephalin,polyhydric alcohol esters, fatty alcohol esters, hydrophilic lanolinderivatives, and hydrophilic beeswax derivatives.

Alternatively, the NO donor(s) can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration, which is also preferred for thepractice of the present invention. Such carriers enable the compounds ofthe invention to be formulated in dosage forms such as tablets, pills,capsules, liquids, gels, syrups, slurries, suspensions and the like, forbuccal or sublingual administration or oral ingestion by a patient to betreated. These carriers may be selected from sugars, starches, celluloseand its derivatives, malt, gelatine, talc, calcium sulphate, vegetableoils, synthetic oils, polyols, alginic acid, phosphate bufferedsolutions, emulsifiers, isotonic saline, and pyrogen-free water.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for buccal, sublingual or oral use can beobtained by combining the active compounds with solid excipients,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable auxiliaries, if desired, to obtaintablets or dragee cores. Suitable excipients are, in particular, fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;cellulose preparations such as, for example, maize starch, wheat starch,rice starch, potato starch, gelatine, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Such compositions may beprepared by any of the methods of pharmacy but all methods include thestep of bringing into association one or more therapeutic agents asdescribed above with the carrier which constitutes one or more necessaryingredients. In general, the pharmaceutical compositions of the presentinvention may be manufactured in a manner that is itself known, e.g., bymeans of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterisedifferent combinations of active compound doses.

For buccal or sublingual administration, the formulations of theinvention can be provided in the form of a tablet, patch, troche, or infree form, such as a gel, ointment, cream, or gum. Examples of suitablebuccal or sublingual formulations and devices are disclosed, forexample, in U.S. Pat. Nos. 5,863,555, 5,849,322, 5,766,620, 5,516,523,5,346,701, 4,983,395, and 4,849,224. Such formulations and devices canuse a suitable adhesive to maintain the device in contact with thebuccal mucosa. Examples of suitable adhesives are found, for example, inU.S. Pat. Nos. 3,972,995, 4,259,314, 4,680,323; 4,740,365, 4,573,996,4,292,299, 4,715,369, 4,876,092, 4,855,142, 4,250,163, 4,226,848, and4,948,580. Typically, the adhesive comprises a matrix of a hydrophilic,e.g., water soluble or swellable, polymer or mixture of polymers thatcan adhere to a wet, mucous surface. These adhesives can be formulatedas ointments, thin films, tablets, troches, and other forms. Othernon-limiting buccal or sublingual formulations are disclosed in U.S.Pat. Nos. 7,067,116; 7,025,983; 6,923,981; 6,596,298; 6,726,928;6,709,669; 6,509,040; 6,413,549; 5,976,577; 5,827,541; 5,738,875;5,648,093; 5,631,023; 5,188,825; 4,020,558; 4,229,447; 3,972,995;3,870,790; 3,444,858; 2,698,822; 3,632,743, and U.S. PublishedApplication Nos. 20070059361; 20040247648; 20040131661; and 20040028730.In some embodiments, the dosage forms are prepared usingpharmaceutically acceptable excipients. Illustrative excipients that arecommonly formulated into buccal and sublingual dosage forms includemaltodextrin, colloidal silicon dioxide, starch, starch syrup, sugar andα-lactose.

Pharmaceuticals which can be used orally include push-fit capsules madeof gelatine, as well as soft, sealed capsules made of gelatine and aplasticizer, such as glycerol or sorbitol. The push-fit capsules cancontain the active ingredients in admixture with filler such as lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added.

Dosage forms of the NO donors may also include injecting or implantingcontrolled releasing devices designed specifically for this purpose orother forms of implants modified to act additionally in this fashion.Controlled release of an active compound of the invention may beachieved by coating the same, for example, with hydrophobic polymersincluding acrylic resins, waxes, higher aliphatic alcohols, polylacticand polyglycolic acids and certain cellulose derivatives such ashydroxypropylmethyl cellulose. In addition, controlled release may beachieved by using other polymer matrices, liposomes and/or microspheres.

The NO donors may be administered over a period of hours, days, weeks,or months, depending on several factors, including the severity of theneuropathic condition being treated, whether a recurrence of thecondition is considered likely, etc. The administration may be constant,e.g., constant infusion over a period of hours, days, weeks, months,etc. Alternatively, the administration may be intermittent, e.g., activecompounds may be administered once a day over a period of days, once anhour over a period of hours, or any other such schedule as deemedsuitable.

The compositions of the present invention may also be administered tothe respiratory tract as a nasal or pulmonary inhalation aerosol orsolution for a nebulizer, or as a microfine powder for insufflation,alone or in combination with an inert carrier such as lactose, or withother pharmaceutically acceptable excipients. In such a case, theparticles of the formulation may advantageously have diameters of lessthan 50 micrometers, suitably less than 10 micrometers.

In some embodiments, the NO donor may be administered in a compositionwith another treatment for neuropathic pain. In some embodiments, theother treatment for neuropathic pain is not an opioid analgesic such asmorphine. In some embodiments, the other treatment for neuropathic painis selected from one or more of anticonvulsants such as carbamazepine,gabapentin, phenytoin, pregabalin and valproate; antidepressants such asamitriptyline, desipramine and duloxetine; central α-2 adrenergicagonists such as clonidine and tizanidine; corticosteroids such asdexamethasone and prednisone; NMDA-receptor antagonists such as memantinand dextromethorphan, oral sodium channel blockers such as mexiletine ortopical compositions such as capsaicin, EMLA® and lidocaine; or othercompositions including baclofen and pamidronate. In some embodiments,particularly when the NO donor is a furoxan NO donor of formula (I), theNO donor is administered in a composition with an opioid analgesic,especially a μ-opioid analgesic such as morphine.

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the instantapplication.

Throughout the specification the aim has been to describe the preferredembodiments of the invention without limiting the invention to any oneembodiment or specific collection of features. Those of skill in the artwill therefore appreciate that, in light of the instant disclosure,various modifications and changes can be made in the particularembodiments exemplified without departing from the scope of the presentinvention. All such modifications and changes are intended to beincluded within the scope of the appended claims.

In order that the invention may be readily understood and put intopractical effect, particular preferred embodiments will now be describedby way of the following non-limiting examples.

EXAMPLES Example 1

Anti-Allodynic Efficacy and Potency of the NO Donor Compound 1

Preparation of Compound 1

Compound 1 was prepared by the method a set out in Journal ofHeterocyclic Chemistry, 1989, 26(5): 1345-1347. But-2-ene aldehyde wastreated with sodium nitrite in the presence of aqueous acetic acid toprovide Compound 1.

Experimental Animals

Adult male Wistar rats (225 g-250 g) were purchased from the CentralAnimal Breeding House, The University of Queensland (Brisbane,Australia) and were housed in a temperature-controlled environment(21±2° C.) with a 12 h/12 h dark/light cycle; food and water wereavailable ad libitum. Rats were given at least a 24 h acclimatisationperiod prior to experimentation. Ethics approval for these experimentswas obtained from the Animal Experimentation Ethics Committee of TheUniversity of Queensland.

Drugs and Materials

Compound 1 was synthesized by Dr Craig Williams, School of Chemistry andMolecular Biosciences, The University of Queensland. Streptozotocin,citric acid, and trisodium citrate were purchased from Sigma-Aldrich PtyLtd (Sydney, Australia). Morphine hydrochloride powder and vialscontaining sodium benzylpenicillin (Benpen™) vials containing 600 mg ofpowder were purchased from the Central Pharmacy of the Royal Brisbaneand Womens Hospital (Brisbane, Australia). Morphine sulfate ampouleswere obtained from Hameln Pharmaceuticals GmbH (Hameln, Germany).Xylazine (Ilium Xylazil™), Zoletil 100® solution (containing: tiletamineHCl 2.5 mg/mL and zolazepam HCL 2.5 mg/mL), sodium benzylpenicillin(Benpen™) vials containing 600 mg of powder and topical antibioticpowder (containing: neomycin sulphate 2.5 mg, sulfacetamide sodium 100mg, nitrofurazone 2 mg, phenylmercuric nitrate 0.05 mg and benzocaine 5mg in 50 g soluble powder) were purchased from Provet Queensland Pty Ltd(Brisbane, Australia). Blood glucose meters (Precision QID, Accucheckadvantage 2) and glucose testing electrodes (Precision Plus™ andAccucheck Advantage™) were purchased from the Campus Pharmacy at TheUniversity of Queensland (Brisbane, Australia). Medical grade CO₂ and O₂were purchased from BOC gases Ltd (Brisbane, Australia) and isoflurane(Isoflo™) was purchased from Abbott Australasia Pty Ltd (Sydney,Australia).

Test and Control Articles

Stock solutions of morphine hydrochloride (26.6 mM) were prepared indistilled water (concentrations expressed as the free base). A stocksolution (24 μM) of Compound 1 and subsequent dilutions were prepared ina mixture of DMSO: distilled water (90%:10%). Compound 1 and morphinehydrochloride stock solutions were protected from light and stored at2-4° C. until required.

Diabetes Induction

Whilst anaesthetized with a mixture of Zoletil 100® (tiletamine HCl0.625 mg and zolazepam 0.625 mg) and xyalazine HCl (5 mg) administeredby intraperitoneal injection to induce deep and stable anaesthesia,adult male Wistar rats were administered single intravenous bolus dosesof streptozotocin (STZ, 75 mg/kg) via a cannula surgically inserted intothe superior aspect of the bifurcation of the internal and externaljugular veins. Following this, the jugular cannula was withdrawn, thevein tied off, and the incision sutured. Following surgery, ratsreceived antibiotic prophylaxis in the form of topical dusting powderover the sutured incisions, as well as a subcutaneous injection ofbenzylpenicillin (60 mg). Rats were placed in individual cages, keptwarm and monitored closely during the surgical recovery period. Ratswere classified as diabetic if on day 10 post-STZ administration, theirdaily water intake was ≥100 mL and their corresponding blood glucoselevels (BGLs) were ≥15 mM.

Development of Tactile (Mechanical) Allodynia

Calibrated von Frey filaments were used to document the time course forthe development and maintenance of tactile allodynia (defining symptomof PDN). Tactile allodynia was considered to be fully developed when vonFrey paw withdrawal thresholds in the hindpaws of STZ-diabetic rats were≤6 g compared with ˜12 g in the same animals prior to the induction ofdiabetes with STZ.

Test and Control Articles: Antinociceptive Testing

In this present study, the antinociceptive effects of single s.c. bolusdose of Compound 1 and morphine administered to control non-diabeticWistar rats were defined as “Week 0” and these effects were comparedwith the corresponding effects produced in STZ-diabetic Wistar ratstested at 10, 14 and 24 weeks post-STZ administration.

Briefly, baseline PWTs were determined for both hindpaws prior to eachantinociceptive testing session in the non-diabetic control group atweek 0 as well as in groups of STZ-diabetic rats at 10, 14 and 24 wks.Following administration of single subcutaneous (s.c.) bolus doses ofthe test (Compound 1) or control (morphine, vehicle) articles to groupsof STZ-diabetic rats, PWTs were determined at the following post-dosingtimes: 0.25, 0:5, 0.75, 1, 1.25, 1.5, 2 and 3 h.

Tactile (mechanical) allodynia was fully developed in the hindpaws ofSTZ-diabetic Wistar rats by ˜9-10 wks post-STZ administration (FIG. 1).

The PWT vs time data were normalized by subtracting the pre-dosingbaseline PWT values for each individual rat to obtain ΔPWT values asfollows: Normalized (Δ)PWT value=Post-treatment PWT value−PWT valueprior to treatment.

ΔPWT versus time curves were then constructed. Trapezoidal integrationwas used to estimate ΔPWT AUC values for individual rats in thenon-diabetic and STZ-diabetic rat treatment groups. ΔPWT AUC versus dosecurves were constructed and GraphPad™ Prism was used to estimate the˜ED₅₀ values.

Temporal Development of Morphine Hyposensitivity in STZ-Diabetic Rats

Single s.c. bolus doses of morphine (1.8-21.4 μmol/kg s.c.) produceddose-dependent antinociception in the hindpaws of control non-diabeticrats (FIG. 2A) as well as dose-dependent anti-allodynia in the hindpawsof STZ-diabetic rats (FIG. 2 B-D). The peak effect was observed at˜30-60 min post-dosing with a corresponding duration of action of ˜2-3 h(FIG. 2). At week 10 post-STZ administration (FIG. 2B respectively),single bolus doses of morphine at 2.8 μmol/kg fully reversed mechanicalallodynia at the time of peak response such that the mean (±SEM) PWTvalue (10.7±0.6 g) matched that for the hindpaws of non-diabetic rats.By week 14 post-STZ administration, single bolus doses of morphine at1.8 μmol/kg were no longer effective and the anti-allodynic potency ofmorphine at 2.8 μmol/kg was significantly reduced (FIG. 2C). However,increasing the dose of morphine 4-fold to 10.5 μmol/kg at week 14post-STZ administration completely relieved mechanical allodynia in thehindpaws at the time of peak response (FIG. 2C). By week 24 post-STZadministration, the anti-allodynic potency of single s.c. bolus doses ofmorphine at 10.5 and 21.4 μmol/kg was markedly reduced (FIG. 2D).

These results show the temporal decrease in the potency of morphineadministered as single s.c. bolus doses at 10-, 14- and 24-wks post-STZadministration. Specifically, by week 24 post STZ-administration, themean (±SEM) extent and duration of anti-allodynia (ΔPWT AUC values)produced by the largest dose of morphine administered (21.4 μmol/kg) wassimilar to that produced by the 10.5 μmol/kg bolus dose of morphine at14 weeks post-STZ, indicative of marked morphine hyposensitivity inthese animals. Hence, it is clear that morphine hyposensitivitydeveloped in a temporal manner as PDN progressed in these STZ-diabeticrats.

Temporal Changes in the Anti-Allodynic Potency of Compound 1 inSTZ-Diabetic Rats

Single s.c. bolus doses of Compound 1 (2-20 fmol/kg s.c.) produceddose-dependent antinociception in the hindpaws of control non-diabeticrats (FIG. 3A). Administration of single s.c. bolus doses of Compound 1at 10-, 14- and 24-wks post-STZ administration produced dose-dependentanti-allodynia/antinociception in the rat hindpaws (FIG. 3 B-D) but thedoses required at 14- and 24-wks (8-800 pmol/kg) were ˜10,000-foldhigher than the effective doses at 10-wks post-STZ administration (8-800fmol/kg). Specifically, at 10 wks post-STZ administration, the 8 fmol/kgdose produced complete relief of mechanical allodynia at the time ofpeak effect (FIG. 3B). Peak anti-allodynic responses (PWT: 10.6±0.5 g)were observed at ˜75 min post-dosing and the corresponding meandurations of action were >3 h (FIG. 3B). By 14 weeks post-STZadministration (FIG. 3C), the doses of Compound 1 that had been usedsuccessfully at week 10 post-STZ administration to fully reversemechanical allodynia in the hindpaws, were no longer effective. However,increasing the magnitude of the s.c. bolus doses of Compound 1 to 8, 80and 800 pmol/kg (FIG. 3C), again resulted in dose-dependentanti-allodynia in these animals. Following administration of single s.cbolus doses of Compound 1 at 80 and 800 pmol/kg, mechanical allodynia inthe hindpaws of STZ-diabetic rats at 14- (FIG. 3C) and 24-wks (FIG. 3D)post-STZ administration was fully alleviated at the time of peakresponse such that the mean (+SEM) peak PWT values were similar tobaseline PWT values for control non-diabetic rats and the correspondingduration of action was ≥3 h.

These results show the large decrease in the potency of single s.c.bolus doses of Compound 1 at 14 and 24 wks c.f. 10-wks post-STZadministration. Specifically, by week 24 post STZ-administration, themean (±SEM) extent and duration of anti-allodynia (ΔPWT AUC value)produced by the largest dose of Compound 1 administered (800 pmol/kg)was similar to that produced by the 8 fmol/kg bolus dose of Compound 1at 10-wks post-STZ in these animals. At 24 weeks post-STZadministration, the bolus doses of Compound 1 that were effective at 14weeks post-STZ administration continued to evoke dose-dependentanti-allodynia with only a small decrease in ΔPWT AUC values relative tothe corresponding values determined at week 14 post-STZ administration.

Example 2

Effects of the NO Donor Compound 1 on Systolic Blood Pressure inNormotensive Wistar Rats

Experimental Animals

Adult male Wistar rats (300 g-350 g) were purchased from UQBR(University of Queensland Biological Resources), The University ofQueensland (Brisbane, Australia) and were housed in atemperature-controlled environment (21±2° C.) with a 12 h/12 hdark/light cycle; food and water were available ad libitum. Rats weregiven at least a 24 h acclimatisation period prior to experimentation.Ethics approval for these experiments was obtained from the AnimalExperimentation Ethics Committee of The University of Queensland.

Drugs and Materials

Compound 1 was synthesized by Dr Craig Williams, School of Chemistry andMolecular Biosciences, The University of Queensland. Zoletil 100®solution (containing: tiletamine HCl 2.5 mg/ml and zolazepam HCL 2.5mg/mL), was purchased from Provet Queensland Pty Ltd (Brisbane,Australia).

Blood Pressure Measurement

Sedation was induced by the intraperitoneal administration of Zoletil®(tiletamine 15 mg kg⁻¹ and zolazepam 15 mg kg⁻¹). Using a tail pulsetransducer (MLT1010) and an inflatable tail cuff with a Capto SP844physiological pressure transducer (MLT844/D), systolic blood pressurewas recorded via a PowerLab data acquisition unit (ADInstruments,Sydney, Australia). Tails were warmed with lukewarm water prior to eachblood pressure reading to increase peripheral circulation so as tofacilitate blood pressure recordings using the tail cuff method.

This study was designed to assess the effects of single s.c. bolus dosesof the furoxan NO donor, Compound 1, relative to vehicle (DMSO:water,90:10) on systolic blood pressure in normotensive, non-diabetic, adultmale Wistar rats. Single bolus doses of Compound 1 were administered bys.c. injection into the back of the neck of adult male normotensive,non-diabetic Wistar rats in the dose range, 80 fmol/kg to 80 μmol/kg.Specifically, the doses of Compound 1 tested were 80 fmol/kg (n=3), 800pmol/kg (n=8), 8 nmol/kg (n=6), 800 nmol/kg (n=4), 8 pmol/kg (n=4), 80μmol/kg (n=3).

Following administration of single s.c. bolus doses of the furoxan NOdonor, Compound 1, to normotensive non-diabetic adult male Wistar rats,there were no significant effects (p>0.05) on systolic blood pressurefor doses in the range, 80 fmol/kg to 8 μmol/kg (FIG. 4). However, atthe highest dose tested (80 μmol/kg, n=3), mean (±SEM) systolic bloodpressure decreased significantly (p<0.05) from 111.3 (±1.5) mm Hg justprior to administration of Compound 1 (80 μmol/kg) to 79.8 (±0.5) mm Hgat the time of peak effect at 30 min post-dosing (FIG. 4). By 1 hpost-dosing, mean systolic blood pressure in the same animals (112.6±0.4mm Hg) did not differ significantly (p>0.05) from baseline systolicblood pressure determined just prior to dose administration (FIG. 4).

Example 3

Compound 1 Evokes Anti-Allodynia in STZ-Diabetic Wistar Rats: Non-OpioidMechanism and Partial Activation of sGC

Experimental Animals

Adult male Wistar rats were used as for Example 1 except that they werepurchased from UQBR (University of Queensland Biological Resources).

Drugs and Materials

Compound 1 was synthesized by Dr Craig Williams, School of Chemistry andMolecular Biosciences, The University of Queensland (Brisbane,Australia). 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) andnaloxone hydrochloride dihydrate were purchased from Sigma-Aldrich PtyLtd (Sydney, Australia). Stock solutions of naloxone hydrochloride (30.6mM) were prepared in distilled water (concentrations expressed as thefree base). Fresh solutions of ODQ (53.4 mM) in DMSO were prepared foreach experiment.

Method

Allodynia was assessed using calibrated von Frey filaments as describedin Example 1.

As most studies indicate that the biological effects of NO are mediatedprimarily via activation of soluble guanylyl cyclase (sGC) to increasecellular levels of cGMP, the contribution of NO/sGC/cGMP signaling tothe anti-allodynic effects of Compound 1 was assessed. Briefly, 14-18week STZ diabetic rats were pre-treated with a single bolus dose of ODQ(53.4 μmol·kg s.c.), a selective inhibitor of sGC at 60 min prior toadministration of single bolus doses of Compound 1 (800 pmol/kg s.c.).Von Frey PWTs in the hindpaws were determined prior to ODQadministration and at the following times post-ODQ administration, 0.5,1 hour (immediately prior to Compound 1 dosing), 1.25, 1.5, 1.75, 2,2.5, 3, 3.5 and 4 hours.

To examine the possible involvement of opioid receptors in mediating theanti-allodynic/antinociceptive effects of Compound 1, the non-specificopioid receptor antagonist, naloxone, was employed. Briefly, single s.c.bolus doses of the non-selective opioid antagonist, naloxone (1.25μmol/kg), were administered 10 minutes prior to single s.c. bolus dosesof Compound 1 (800 pmol/kg) to 14-18 week STZ-diabetic rats. Forcomparative purposes, naloxone-pretreated 14-18 week STZ-diabeticreceived single s.c. bolus doses of morphine (7 μmol/kg) instead ofCompound 1.

Compound 1 Evokes Anti-Allodynia in STZ-Diabetic Wistar Rats: Non-OpioidMechanism

Pre-treatment of 14-18 wk STZ-diabetic rats with single bolus doses ofnaloxone (1.25 μmol/kg s.c.) 10 min before administration of singlebolus doses of Compound 1 (800 pmol/kg s.c.) did not significantlyattenuate (P>0.05) the extent and duration of anti-allodynia (ΔPWT AUCvalues) compared with vehicle-pretreated STZ-diabetic rats administeredbolus doses of Compound 1 at 800 pmol/kg (FIG. 5A) such that the mean(±SEM) ΔPWT AUC values did not differ significantly (P>0.05) between thetwo groups. Importantly, pre-treatment of 14-wk STZ-diabetic rats withnaloxone (1.25 μmol/kg s.c.) 10 min prior to single bolus doses ofmorphine at 7 μmol/kg s.c., completely abolished the anti-allodyniceffects of morphine relative to vehicle pre-treated STZ-diabetic ratsadministered the same dose of morphine (FIG. 5B). Together, thesefindings indicate that although the anti-allodynic effects of theprototypic μ-opioid agonist, morphine, were opioid-receptor mediated,the anti-allodynic effects of Compound 1 in STZ-diabetic rats were not.

Compound 1 Anti-Allodynia in STZ-Diabetic Wistar Rats: PartialActivation of SGC

Pre-treatment of 16-wk STZ-diabetic rats with a single bolus dose of ODQ(53.4 μmol/kg s.c.) 60 min prior to a single bolus dose of Compound 1(800 pmol/kg s.c.) produced a transient increase in mean PWTs in thehindpaws which returned to baseline by 5 min post-dosing. Although ODQdid not significantly attenuate the anti-allodynic effects of Compound 1(800 pmol/kg) in the first 45 minutes after Compound 1 administration,the anti-allodynic effects of Compound 1 (800 pmol/kg) weresignificantly attenuated from 45 minutes onwards (FIG. 5C) such that themean (±SEM) ΔPWT AUC was significantly (P<0.05) less than thecorresponding value for vehicle-pretreated STZ-diabetic ratsadministered the same dose of Compound 1. These findings suggest thatthe anti-allodynic effects of Compound 1 in STZ-diabetic rats aremediated by both cGMP-dependent and independent mechanisms.

Example 4

Absence of Behavioural Side-Effects with Compound 1 in Contrast toMorphine

Even at the highest dose of Compound 1 (800 pmol/kg s.c.) administeredto STZ-diabetic rats, there were no discernable side-effects. This wasin contrast to morphine which produced staring and sedation at thehighest dose tested (21 μmol/kg).

Example 5

Effects of the Furoxan NO Donor Compound 1, Morphine or Vehicle onForskolin-Stimulated cAMP Responses in HEK293 Cells Transfected withOpioid Receptors

The cellular mechanism of action by which Compound 1 evokes itsanti-allodynic effects was investigated using HEK293 cells stablytransfected with mouse μ-, δ- or κ-opioid receptors (MOP, DOP and KOPreceptors respectively). Non-transfected HEK293 cells were used as acontrol. Transfected HEK293 cells were maintained in Dulbecco's ModifiedEagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) andgeneticin (1 mg/mL). Non-transfected HEK293 cells were maintained inDMEM supplemented with 10% FBS.

The functional interaction between opioids and opioid-like compounds andspecific opioid G-protein-coupled receptors (GPCRs) was assessed bymeasuring changes in intracellular cAMP levels relative to basal levels.The Alphascreen cAMP functional assay measures agonist and antagonistactivity of GPCRs by stimulating cells to either increase or decreaseintracellular cAMP levels. The assay was performed using an AlphascreencAMP kit according to the manufacturer's instructions.

The assay was performed in a 384 well plate and each data point wasperformed in triplicate. Compound 1 was analyzed on the same plate ascontrols and cAMP standards.

Detection of cAMP is based on the competition between intracellular cAMPand biotinylated cAMP linked streptavidin-coated donor beads foranti-cAMP conjugated acceptor beads. When the donor and acceptor beadsare in close proximity visible light emitted at 520-620 nm is detectedusing the Envision multilabel plate reader.

Stocks Solution Preparation

500 mM IBMX Solution

100 mg IBMX was dissolved in 900 μL DMSO to give a 500 mM stocksolution. Aliquots were stored at −20° C.

50 mM Forskolin Solution

5 mg forskolin was dissolved in 244 μL of 95% ethanol to give a 50 mMstock solution and was stored at −20° C. and used as required.

Fresh Reagents

The following reagents were prepared fresh in 50 mL conical tubes:

Stimulation Buffer (1×HBSS, 0.1% BSA, 1 mM IBMX)

For 50 mL, 5 mL 10×HBSS was added to a 50 mL tube and made up to 50 mLwith water. 50 mg BSA was added and the solution allowed to stand at 37°C. until BSA was dissolved then 100 μL of IBMX was added while thebuffer was at 37° C. to ensure that the IBMX did not precipitate.

Lysis Buffer (0.3% Tween-20, 5 mM Hepes, 0.1% BSA)

For 40 mL, 1.2 mL 10% Tween-20 and 200 μL 1M HEPES were added to a 50 mLtube and made up to 40 mL with water. 50 mg BSA was added and thesolution allowed to stand at 37° C. until BSA was dissolved.

Stimulation Buffer with Forskolin (*200 μM in Stimulation Buffer)

Forskolin was made up to a dilution of 1:250 from the 50 mM stock andadded to the required amount of stimulation buffer. It should be notedthat the final concentration in the assay plate will be halved.

Preparation of Compounds

Compound 1

Stock solutions of Compound 1 at 1 mM and 100 μM were prepared in anappropriate diluent and stored at 4° C. Compound 1 was diluted freshfrom stock solutions to a working concentration in stimulation bufferwith forskolin. It should be noted that as compounds are diluted 1:1with cells in the assay, the working concentrations are twice the finalrequired concentration.

Control Compounds

Compounds with known activity against MOP, DOP and KOP receptorsincluding morphine, DPDPE and U69,593, respectively were used as controlopioid agonists. Control opioid agonists were used at 1 μM and werediluted fresh in stimulation buffer with forskolin from stocks stored at−20° C. For the assay 5 μL of prepared control compounds were added perwell in triplicate.

Preparation of Cells

The assay performance was optimized by using cells at low passage numberand at 70-90% confluence. The cells were prepared for the assay byremoving growth medium, adding Versene and then incubating at 37° C. forapproximately 5 minutes to allow cells to detach from the tissue cultureplastic. The cells were collected and centrifuged for 2 minutes at275×g. The supernatant was decanted and the cell pellet resuspended in1×PBS. The cell concentration was determined using a haemocytometer. Thecells were re-centrifuged for 2 minutes at 275×g and the supernatantdecanted. The cells were resuspended in stimulation buffer to a finalconcentration of 1-4×10⁴ cells per mL. Note that the cell number willinfluence the cAMP levels measured before (basal) and after adenylylcyclase activation. A cell titration was performed to optimize thedifference between basal and stimulation levels of cAMP. Cells wereincubated in stimulation buffer for 20 to 30 minutes at 37° C. prior toadding 5 μL to wells containing test and control compounds. Note thatcells were not added to the cAMP standards.

Preparation of a cAMP Standard Curve

A standard cAMP dilution series was prepared from the kit supplied using50 μM cAMP solution. The solution was vortexed at maximum intensity for5 seconds before use then serially diluted to provide a finalconcentration range from 5 μM to 0.5 nM cAMP (for example: 5 μM, 0.5 μM,50 nM, 15 nM, 5 nM, 1.5 nM, and 0.5 nM cAMP). A positive control (nocAMP) was also included. For the assay 10 μL of standard dilutions wereadded per well in triplicate.

Preparation of Acceptor and Donor Bead Solutions

The anti-cAMP conjugated acceptor beads and streptavidin-coated donorbeads are light sensitive and need to be handled under subdued lightingor under lights fitted with green filters. Once the beads have beenadded to the assay plate it should be wrapped in foil so thatincubations are performed in the dark. Acceptor and donor bead solutionswere prepared in 15 mL conical tubes while the cells were incubating andkept in the dark until required. For the acceptor bead solution, 10 μLbead suspension per mL of lysis buffer was gently mixed. For the donorbead solution 10 μL bead suspension per mL of lysis buffer and 0.75μL/mL of cAMP-biotin were used and mixed gently.

cAMP Assay Procedure

-   -   1. Standards (10 μL/well), control compounds (5 μL/well) and        Compound 1 (5 μL/well) were added into 384-well plates and        sealed with Top Seal adhesive sealing film and left at room        temperature until the cell incubation was complete.    -   2. 5 μl aliquots of cells incubated in stimulation buffer was        added to the wells containing Compound 1 and control compounds,        but not to the standards. The cells and compounds were incubated        for 30 min at 37° C.    -   3. 10 μL lysis buffer was added per well.    -   4. Under subdued lighting 5 μL acceptor bead solution was added        to each well. The plate was wrapped in foil and incubated at        room temperature with gentle mixing on an orbital shaker for 60        min.    -   5. Also under subdued lighting 5 μL donor bead solution was        added to each well, the plate wrapped in foil, and then        incubated overnight at room temperature with gentle mixing on an        orbital shaker.    -   6. cAMP levels were measured using an Envision multilabel plate        reader.        Results Analysis

The results were analyzed using the Graphpad PRISM® software program tocalculate the intracellular levels of cAMP for each triplicate datapoint and the standard deviation of these data points.

In some assays, cells underwent pre-treatment with pertussis toxin (PTX)(100 ng/mL for 16 hours at 37° C.) or naloxone (10 μM for 30 minutes at37° C.) or ODQ (10 μM for 30 minutes at 37° C.).

The results are shown in FIGS. 6A-C, 7 and 8.

Compound 1 did not appear to inhibit forskolin-stimulated cAMP formationin HEK293 cells transfected with δ-opioid receptors (DOP receptors)(FIG. 6A) or κ-opioid receptors (KOP receptors) (FIG. 6B) or innon-transfected HEK293 cells (FIG. 6C) across the entire concentrationrange tested. The standard compounds used were [D-Pen^(2,5)]-Enkephalinhydrate (DPDPE, DOP transfected cells),N-methyl-2-phenyl-N-[(5R,7S,8S)-7-pyrrolidin-1-yl-1-oxaspiro[4,5]dec-8-yl]acetamide(U69,593, KOP transfected cells) and morphine (non-transfected cells) asagonists at DOP, KOP and MOP receptors respectively.

However, Compound 1 inhibited forskolin-stimulated cAMP formation inHEK293 cells transfected with μ-opioid receptors (MOP receptors) at mM,pM and aM concentrations in an analogous manner to morphine (FIG. 7).

The inhibition of forskolin-stimulated cAMP formation in MOP-transfectedHEK293 cells produced by 1 aM of Compound 1 was abolished bypre-incubation with pertussis toxin but not by naloxone. In contrast,the inhibition of forskolin-stimulated cAMP formation in MOP-transfectedHEK293 cells by 1 μM morphine was blocked by pre-incubation with bothpertussis toxin and naloxone (FIG. 8).

It was found that pM-μM concentrations of morphine inhibitedforskolin-stimulated cAMP formation in a concentration-dependent mannerwith even lower concentrations of morphine producing superactivation ofcAMP responses (FIG. 9).

Example 6

Displacement of Specific Binding of [3H]-DAMGO from Membranes ofMOP-Transfected HEK293 Cells

A binding assay was performed to determine whether Compound 1 binds atthe MOP receptor.

Stably transfected MOP-HEK cells were plated in a 3×10 cm dish andwashed with (3×5 mL) 50 mM Tris-HCl (pH 7.4) when sufficientlyconfluent. The cells were scraped off the dish in 1.5 mL 50 mM Tris-HCl(pH 7.4) using a transfer pipette and place in 15 mL falcon tubes. Thecells were sonicated for a 5 second burst and placed on ice. The proteinconcentration was estimated. The cells were transferred to a 1.5 mLeppendorf tube and centrifuged at 18,000 rcf for 30 minutes at 4° C. Thesupernatant was removed and the cells resuspended in 50 mM Tris-HCl (pH7.4) and 1 mg/mL BSA. An aliquot containing 30-50 μg/200 μL protein wastaken and [³H]DAMGO was added (200 μL in Tris-HCl, pH 7.4) with varyingconcentrations of unlabelled [D-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalinacetate (DAMGO) or Compound 1 (200 μL in Tris-HCl, pH 7.4), total volume600 μL. The solution was incubated for 1 hour at room temperature withslow rotation.

The results are shown in FIG. 10 and show that Compound 1 does not bindsignificantly to MOP receptors.

Example 7

Effects of Compound 1 on cGMP Production

For assessment of cGMP levels in MOP-transfected HEK cells, the methodsused were similar to those described in Example 6 with the exceptionthat forskolin was omitted from the incubations. cGMP levels werequantified using AlphaScreen detection kits according to themanufacturer's instructions. Briefly, 5 μL aliquots of the cellsuspension were added to individual wells containing 5 μL aliquots oftest or control article solutions at the concentration of interest intriplicates in 384-well plates and incubated for 30 min at 37° C. cGMPstandard solutions were serially diluted in stimulation buffer to giveconcentrations in the range of 5×10⁻¹⁰-5×10⁻⁶ M and were added at 10μL/well. After cells were lysed with lysis buffer, 5 μL aliquots ofacceptor bead mix (10 μl/mL of acceptor beads and 1/3000 anti-cGMPantibody for cGMP detection in lysis buffer) were added to each well.After plates were incubated in the dark at room temperature for 60 min,5 μL aliquots of donor bead mix (10 μL/mL of donor beads and 0.3 nMbiotinylated cGMP in lysis buffer) were added per well and plates wereincubated for 16 h in the dark at room temperature. Bioluminescence wasmeasured using an Envision Multilabel Plate Reader (PerkinElmer LifeSciences).

Lack of Involvement of Guanylyl Cyclase in Effects of Compound 1 onCellular cAMP Responses

Pre-incubation of HEK-MOP cells with the sGC inhibitor, ODQ (10 μM), didnot significantly attenuate the modulatory effects of Compound 1 onforskolin-stimulated cAMP formation (FIG. 8). As the cell membranepermeable cGMP analogue 8-Br-cGMP (FIG. 11, 8-Br-cGMP) did not inhibitforskolin-stimulated cAMP responses, the contribution of NO/sGC/cGMPsignalling to the modulatory effects of Compound 1 onforskolin-stimulated cAMP formation, is further discounted.

Example 8

Role of Lipid Rafts/Caveolae in Modulation of Cellular cAMP Responseswith Compound 1

Materials and Methods

Compound 1 was synthesised by Dr Craig Williams, School of Chemistry andMolecular Biosciences, The University of Queensland, Brisbane,Australia. (2-Hydroxypropyl)-β-cyclodextrin (βCD) and chlolesterol(water soluble) were purchased from Sigma-Aldrich Pty Ltd (Sydney,Australia). Dulbecco's Modified Eagle Medium (DMEM) was purchased fromInvitrogen (Mount Waverley, Australia).

As many signaling proteins including receptors, G-proteins, ion channelsand effectors such as adenylyl cyclase, co-localize within lipidraft/caveolin-rich microdomains in the cell membrane to facilitate rapidsignal transduction cross-talk between molecules (Ostrom Bundy, et al.,J. Biol. Chem., 2004, 279(19):19846-19853), the role of caveolin in theeffects of Compound 1 on forskolin-stimulated cAMP responses in HEK-MOPcells, was assessed. Briefly, HEK-MOP cells were pre-incubated with 2%(2-hydroxypropyl)-β-cyclodextrin (βCD) for 1 h at 37° C. to depletecholesterol from caveloae in the cell membrane. Cells were washed withDMEM and then utilized in the forskolin-stimulated cAMP assay asdescribed above. Following pre-incubation with 2% βCD for 1 h at 37° C.followed by washing with DMEM, additional βCD-treated HEK-MOP cells werethen treated with βCD-cholesterol complexes (10 μg/mL cholesterol-βCD ina 1:6 molar ratio) to replenish cholesterol in caveolae in the cellmembrane (Ostrom, Bundey et al. 2004, J. Biol. Chem.,279(19):19846-19853). Cells were washed with DMEM and then utilized inthe forskolin-stimulated cAMP assay as described above.

Treatment of MOP-transfected HEK293 cells with 2% βCD to deplete lipidrafts/caveolae inhibited the effects of Compound 1 onforskolin-stimulated cAMP responses (FIG. 12), which were reversed upondelivering cholesterol back to the cells with βCD-cholesterol complexes(FIG. 12). These results are consistent with the notion that Compound 1may act, at least in part, via modulation of transduction moleculesresiding in lipid rafts or caveolae domains to regulate downstream cAMPactivity.

Example 9 In Vitro NO Release Profile of Compound 1

The extent of release of NO by the NO donor, Compound 1, was determinedby measuring the concentration of the stable NO metabolite nitrite, (NO₂⁻), using a modified Griess reagent assay involving the measurement ofnitrite after conversion of nitrate to nitrite to give total NO_(x)concentrations in samples. (Miranda, Espey et al. 2001, Nitric OxideBiology and Chemistry, 5(1):62-71).

Briefly, HEK-MOP cells were seeded at 1×10⁵ cells/mL and grown in 10 cmplates to 90% confluence. Four hours prior to experimentation, cellmedium was replaced with phenol red-free DMEM. Cells were treated witheither Compound 1 (1 mM±5 mM L-cysteine) or SIN-1 (1 mM and 10 nM) in 6mL of phenol red-free DMEM and 500 μL samples of the cell-supernatantwere collected prior to addition of the NO donor of interest and at thefollowing times post-treatment: 1, 2, 5, 10, 20, 30, 45, 60, 120, 180and 360 min. Following removal of each supernatant sample, it wasreplaced with 500 μL of phenol red-free DMEM. Samples were immediatelyplaced on ice until NO_(x) concentrations were quantified.

Sample aliquots (100 μL) were added to 96-well plates in duplicates.Nitrate was first converted to nitrite to determine total NO_(x)concentrations with the addition of 100 μL aliquots of vanadium (III)chloride in 1M HCl (800 mg/100 mL) per well, followed by 100 μL aliquotsof modified Griess reagent which converted nitrite to a deep purple azoderivative for spectrophotometric quantification at 560 nm using anEnvision Multilabel Plate Reader (PerkinElmer Life Sciences). A standardcurve was prepared in a similar manner to that used for the knownconcentrations of nitrite (3.125-200 μM) and the line of best fit wasdetermined using linear regression as implemented in the GraphPad Prism™5.0 software program (GraphPad Software). Total NO_(x) and nitriteconcentration was determined through inverse prediction against thenitrite standard curve.

Over a 6-h study period in vitro, the mean (±SEM) amount of NO (asnitrate/nitrite) released by Compound 1 was 1.8% (±2.7) (Table 2). In amanner similar to other NO donors of the furoxan class, co-incubation ofCompound 1 with L-cysteine (50 mM), increased NO_(x) formation by1.5-fold (from 1.8±0.3% to 2.7±0.3%, mean±SEM), likely due to anincrease in thiol-dependent NO release.

TABLE 2 In vitro effects of the NO donor, Compound 1 (in the presenceand absence of L-cysteine) on cGMP activity and extent of NO release (asTotal NOx) over a 6-h study period. cGMP Activity (% of basal Release ofNO Compound cGMP response) (Total NOx %) Compound 1, 1 mM 159 + 20 1.8 +0.3 Compound 1, 1 mM + 114 + 17 N/A ODQ, 10 μM Compound 1, 1 mM + 185 +34 2.7 + 0.3 L-Cysteine 5 mM (1.2 fold increase) (1.5 fold increase)

In HEK-MOP cells, Compound 1 in both the presence and absence ofL-cysteine (5 mM), produced comparatively weak stimulation of cGMPformation in HEK-MOP cells. This finding suggests that the extent towhich Compound 1 stimulates cGMP formation in HEK-MOP cells iscorrelated with the extent to which it releases NO.

Example 10

Comparison of the Development of Tolerance to Morphine and Compound 1Induced Antinociception in Non-Diabetic Control Rats and in STZ-DiabeticRats

For drug-naïve non-diabetic control rats, there was a rapid onset ofantinociception produced by single s.c. bolus doses of Compound 1 at 8fmol/kg (FIG. 13A). Mean (±SEM) peak levels of antinociception occurredat 1 hour post-dosing [PWT: 15.9±0.3 g; ΔPWT: 4.4+(0.4) g] and thecorresponding mean duration of action was −3 hours. After completion ofthe 7-day continuous s.c. infusion of Compound 1 rats were completelytolerant to the antinociceptive effects of Compound 1. After a 2-daywashout period, rats received a second s.c. bolus dose of Compound 1 at8 fmol/kg. Following s.c. administration of the 2^(nd) single s.c. bolusdose of Compound 1, there was a consistent rapid onset ofantinociception and the corresponding mean (±SEM) peak levels ofantinociception appeared to occur 1 hour post-dosing [PWT: 15.6 (±0.7)g; ΔPWT: 4.2 (±0.6) g] with a corresponding mean duration of action of−3 hours. It is clear from visual inspection of FIG. 13A, that theextent and duration of antinociception produced by single s.c. bolusdoses of Compound 1 at 8 fmol/kg did not differ significantly (P>0.05)between that determined in drug naïve and “tolerant” rats.

For drug-naïve non-diabetic control rats administered single s.c. bolusdoses of morphine (2.8 mmol/kg), there was a rapid onset of action withmean (±SEM) peak levels of antiniociception observed at 1 hourpost-dosing [PWT: 16.0 (±0.4) g; Δ PWT: 4.4 (±0.4) g] and a meanduration of action of ˜3 hours (FIG. 13B). Following continuous s.c.infusion of morphine for 7-days, antinociceptive tolerance developedsuch that mean (±SEM) hindpaw PWT values did not differ (P>0.05) frombaseline pre-dosing mean (±SEM) PWT values by 7-days of the infusion(FIG. 13B). After a 2-day washout period, rats received a second s.c.bolus dose of morphine at 2.8 mmol/kg. The corresponding PWT and ΔPWTversus time curves show that these rats were tolerant to theantinociceptive effects of morphine (FIG. 13B; peak PWT: 12 (+0.3) g;ΔPWT: 0.5 (±0.3) g) as insignificant (P>0.05) antinociception wasproduced; [ΔPWT AUC: 0.0 (±0.2) g·h.]. Following administration ofvehicle either by single s.c. bolus or by continuous s.c. infusion for7-days to control non-diabetic rats, there was a complete absence ofantinociception confirming that neither the vehicle nor the experimentalprocedures themselves produce significant antinociception.

Following administration of single s.c. bolus doses of Compound 1 at 800pmol/kg to drug-naïve STZ-diabetic rats, there was a rapid onset ofanti-allodynia (FIG. 14A). Mean (±SEM) peak l evels of anti-allodyniawere observed at 1 h post-dosing [PWT: 11.6 (±0.6) g] and thecorresponding mean duration of action was ˜4 h (FIG. 14A). Aftercompletion of a 7-day s.c. infusion, STZ-diabetic rats were completelytolerant to the anti-allodynic effects of Compound 1. After a 2-daywashout period, STZ-diabetic rats were administered a second s.c. bolusdose of Compound 1 at 800 pmol/kg. Again, there was a rapid onset ofaction with mean (±SEM) peak anti-allodynia observed at ˜1 h post-dosingand a corresponding mean duration of action ˜2-3 h. Comparison of theΔPWT versus time curves shows significant reversal of tolerance in theSTZ-diabetic rat group (FIG. 14 A).

For opioid-naïve STZ-diabetic rats administered single s.c. bolus dosesof morphine (10.4 μmol/kg), there was a rapid onset of anti-allodynia(FIG. 14B). Mean (±SEM) peak levels of anti-allodynia were observed at 1h post-dosing [PWT: 10.7 (±0.3) g]; the corresponding mean duration ofaction was ˜4 h. Following the continuous s.c. infusion of morphine,antinociceptive tolerance developed such that PWT values did not differfrom baseline pre-dosing PWT values by 7-days of the s.c. infusion.After a 2-day washout, rats received a second s.c. bolus dose ofmorphine at 10.5 μmol/kg. The corresponding ΔPWT versus time curves showthat these rats were tolerant to the anti-allodynic effects of morphineas insignificant anti-allodynia was produced in the hindpaws [FIG. 14B;mean (±SEM) peak PWT: 6.1±(0.5) g, mean (±SEM) peak ΔPWT: 1.0±(0.5) g].Following administration of vehicle either by single s.c. bolus or bycontinuous s.c. infusion for 7-days to STZ-diabetic rats, there was acomplete absence of anti-allodynia, confirming that neither the vehiclenor the experimental procedures themselves produce significantanti-allodynia in the hindpaws (FIG. 14C).

Example 11

Synthesis of Furoxan Compounds

Products were purified by column chromatography, preparative thin layerchromatography, filtration of a solid or recrystallisation.

General Synthetic Method A

The appropriate alpha, beta unsaturated aldehyde was treated withsaturated sodium nitrite solution in glacial acetic acid to furnish thedesired aldehyde substituted furoxan. Where appropriate, thisintermediate represented a target structure.

The aldehyde was dissolved in 12 M H₂SO₄ solution and oxidized withaqueous potassium permanganate solution (0.83 eq.) overnight at roomtemperature to produce the acid product which represented an additionaltarget molecule.

The acid was converted to the corresponding acid chloride with thionylchloride in dichloromethane at reflux with catalytic DMF. The resultingacid chloride was treated with ammonia to produce the amide product.

General Synthetic Method B

The appropriate diketone starting material was treated withhydroxylamine hydrochloride in ethanol/water at reflux to furnish a1,2-dioxime. This dioxime was oxidized to the desired furoxan ringstructure via the use of either sodium hypochlorite in the presence ofsodium hydroxide in ethanol at 5° C., or oxidised with chlorine inethanol at room temperature.

In some cases, the diketone starting material was obtained by theoxidation of an appropriate phenylacetic acid by PCC/pyridine indichloromethane at reflux.

General Synthetic Method C

The appropriate aldehyde or ketone starting material was treated withsodium borohydride in methanol at room temperature to provide an allylicalcohol. This alcohol was dissolved in glacial acetic acid and treatedwith aqueous sodium nitrite to provide the furoxan ring structure. Whereappropriate, this represented a target structure.

The hydroxyl furoxan was oxidised to the aldehyde or ketone via the useof activated manganese dioxide in dichloromethane. This represented atarget structure.

The aldehyde containing structures could be further oxidised with Jonesreagent to provide access to the carboxylic acids. These representedfurther target structures.

The carboxylic acid intermediates could be further converted into amidesby treatment with thionyl chloride in DCM at reflux with catalytic DMFto provide the intermediate acid chloride. The acid chloride was treatedwith ammonia in THF to provide the desired amide targets.

General Synthetic Method D

The appropriate 2-aminonitrobenezene was treated with sodiumhypochlorite in the presence of methanol and potassium hydroxide intemperatures ranging from 5° C. to 120° C. This provided the targetfuroxan.

In a slight variation, the 2-aminonitropyridine was treated withiodosobenzene diacetate to produce the desired furoxan ring.

General Synthetic Method E

1,2-diphenyl-1,2,ethanedione or its di-4-fluorophenyl equivalent wastreated with hydroxylamine hydrochloride at 70° C. to generate the monooxime. This intermediate was further treated with hydroxylaminehydrochloride in the presence of sodium hydroxide at room temperature togenerate the dioxime. The dioxime was treated with chlorine to producethe diaryl substituted furoxan target.

General Synthetic Method F

1,3-Cyclopentadione was reacted with sodium nitrite and then withhydroxylamine hydrochloride to generate the 1,2,3-trioxime. Thisintermediate was treated with sodium hypobromide to create the furoxanring structure. The residual oxime functionality was converted back to aketone via treatment with sulphuric acid and formaldehyde to furnish thedesired ketofuroxan.

General Synthetic Method G

Cyclohexanone was treated with ethylnitrite in the presence of sodiumnitrite and acetic acid to generate cyclohex-1,3-dioxime-2-one.Treatment of this with hydroxylamine hydrochloride in methanol providethe trioxime that was then oxidised to the furoxan ring with sodiumhypobromide. The residual oxime was converted back to a ketonefunctionality via treatment with sulphuric acid and formaldehyde.

General Synthetic Method H

The appropriate aromatic aldehyde was treated with(ethyl)triphenylphosphonium iodide to generate the styrene intermediate.Due to its volatility, this alkene was converted directly withoutisolation to the furoxan ring structure via treatment with aqueoussodium nitrite in glacial acetic acid.

General Synthetic Method I

Aniline was treated with trifluoroacetic acid, triphenylphosphine andtriethylamine in carbon tetrachloride to generatephenyl-(trifluoromethyliminochloride). This intermediate was treatedwith diethyl methylphosphonate and butyl lithium at low temperaturefollowed by benzaldehyde. This provided the trifluoromethyl cinnamylketone. This material was then treated according to General Procedure Cwith the exception that the final oxidation was carried out with sodiumhypochlorite in dichloromethane (instead of using activated manganesedioxide). This provided the furoxan target substituted with phenyl andtrifluoromethyl ketone groups.

Table 3 identifies the compounds prepared by each route and providesmass spectral data.

TABLE 3 Compound Synthetic Method Mass spectral data 1 A 151 (MNa⁺), 161(MHCH₃OH⁺) 2 B 141.1 (MH⁺), 163 (MNa⁺) 3 D 137 (MH⁺), 159 (MNa⁺) 4 D 138(MH⁺), 160 (MNa⁺) 5 E 239 (MH⁺), 261 (MNa⁺) 6 A No ions, ¹H nmr showssinglet at 2.30 ppm 7 A 144 (MH⁺), 166 (MNa⁺) 8 A 159 (MH⁺), 181 (MNa⁺)9 A 158 (MH⁺), 180 (MNa⁺) 10 A 173 (MH⁺), 195 (MNa⁺) 11 A 172 (MH⁺), 194(MNa⁺) 12 A 143 (MH⁺), 175 (MHCH₃OH⁺), 197 (MCH₃OHNa⁺) 13 B 177 (MH⁺),199 (MNa⁺) 14 C 213 (MNa⁺), 245 (MCH₃OHNa⁺) 15 C 275 (MCH₃OHNa⁺) 16 F Noions, ¹H NMR shows multiplet centred on 3.36 ppm 17 G 155 (MH⁺), 177(MNa⁺) 18 C No ions. ¹H NMR shows 7.34 (d, 2H), 7.80 (d, 2H), 13.35 (s,1H) 19 D 157 (MH+), 179 (MNa+) (2 amu above theoretical mass) 20 D 167(MH⁺), 189 (MNa⁺) 21 E 297 (MNa⁺) 22 I No ion. ¹H NMR shows 7.61 (m,3H), 7.90 (m, 2H) 23 H 195 (MH⁺), 217 (MNa⁺), 248.9 (MCH₃OHNa⁺) 24 B 207(MH⁺), 229 (MNa⁺), 261 (MCH₃OHNa⁺) 25 C 161.1 (M − CO₂H⁺) 26 C 206(MH⁺), 228 (MNa⁺) 27 C 205 (MH⁺), 227 (MNa⁺) 28 C 263 (MCH₃OHNa⁺) 29 C224 (MH⁺), 245.9 (MNa⁺) 30 C 236 (MH⁺), 258 (MNa⁺) 31 C 193 (MH⁺), 215(MNa⁺) 32 C 223 (MH⁺), 245 (MNa⁺)

Example 12

Synthesis of Compound 33

Compound 33, 5-nitratopentanoic acid, was prepared using the method setout in EP0984012. Briefly, silver nitrate (23.48 g, 0.153 mol) waspre-dried under high vacuum (0.01 mmHg) and subsequently dissolved inanhydrous acetonitrile (70 mL) under an argon atmosphere. The solutionwas warmed to 50° C. and 5-bromovaleric acid (5 g, 0.028 mol), dissolvedin 3 mL of anhydrous acetonitrile, was added quickly by syringe. Aprecipitate formed instantaneously. The mixture was then heated at 80°C. for 20 minutes. On cooling, the precipitate (AgBr) was removed byfiltration. The filtrate was concentrated and the residue partitionedbetween ethyl acetate and water. The ethyl acetate layer was washed withwater, dried (NaSO₄), concentrated and further dried under vacuum (0.01mmHg). ¹H NMR (300 MHz, CDCl₃) δ 1.68 (m, 4H), 2.41 (t, J=7.0 Hz, 2H),4.45 (t, J=4.45 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 20.8, 26.1, 33.2,72.6, 179.3.

Example 13

Effects of Furoxan NO Donors, Compounds 1-32 on Forskolin-StimulatedcAMP Responses in HEK-293 Cells Transfected with μ-Opioid Receptors

The assay described in Example 5 was repeated with Compounds 1-32 toassess the effects of the furoxan NO donors on forskolin-stimulatedcyclic AMP responses in HEK-293 cells transfected with μ-opioid (MOP)receptors. The results are shown in Table 4 expressed as a % offorskolin stimulated cyclic AMP response. In a similar manner tocompound 1 (FIG. 7), these compounds generally produced biphasic effectson forskolin-stimulated cAMP formation in HEK-293 cells transfected withMOP receptors.

TABLE 4 Conc (M) Comp 10⁻¹⁸ 10⁻¹⁷ 10⁻¹⁶ 10⁻¹⁵ 10⁻¹⁴ 10⁻¹³ 10⁻¹² 10⁻¹¹10⁻¹⁰ 10⁻⁹ 10⁻⁸ 10⁻⁷ 10⁻⁶ 10⁻⁵ 10⁻⁴ 10⁻³  5 (n = 3) 68.7  75.8  91.2117.0 105.6 102.3 119.1 76.3  79.2  57.7  73.6 112.7 142.4 120.4  99.8 69.6  6 (n = 3) 60.4  83.9 149.0  79.0^(a)  98.2 128.0  91.1 88.0  61.5 88.2 111.2 110.5 109.7 138.7 115.0  8.1  8 (n = 3) 72.8 115.0 140.5 97.6  94.8 112.7 128.3 88.3  79.4 107.0 114.3 102.9 104.7  90.0  68.2 6.8 13 (n = 3) 61.2 105.3  88.3^(a) 101.5  99.4^(a)  89.6 108.8 66.3106.9  94.9  80.3  78.8  84.5^(a) 109.4^(a)  67.1 40.2 14 (n = 3) 95.1120.5 129.8 103.8 103.4^(a)  94.2  92.8 62.7  69.5 107.4 113.2^(a) 121.5110.6^(a)  51.0^(a)  40.7^(a)  8.5 15 (n = 3) 94.7 107.6 119.5  80.2166.8^(a)  71.5  77.7 57.5 122.0 129.4  88.3 104.5 101.0  84.1  61.6 3.7 16 (n = 3) 62.4^(a) 100.4 108.3  97.7 106.3  98.8  59.3 67.1 101.3125.4  62.2 119.9  83.5 105.2  44.3 18.9 18 (n = 3) 54.7^(a)  94.1  89.6 90.4  85.3  85.4  82.2 77.5  83.6 142.3^(a)  81.7  91.9 101.0  97.7 79.0  7.1 21 (n = 3) 38.7  49.1  67.7  64.0  59.7  61.5  42.9 29.9 38.4  46.5  57.5  47.3  53.0  63.5  42.1  7.1 22 (n = 7) 47.0  59.4^(b) 73.2  89.0 104.6  70.5  62.4 59.2  66.1  65.2  82.1  68.4  87.5  77.9 18.2  0.3 23 (n = 3) 80.2  73.6  77.1  80.4  79.2  87.6  74.7 75.7 56.5  64.0  98.2  89.0  90.0  66.2 121.2 46.6 24 (n = 3) 70.0  70.0 88.0  97.0^(a)  75.1  82.3  90.1 79.4  60.2 132.4  53.8^(a)  94.3  56.7131.1^(a) 106.7^(a) 71.2 27 (n = 3) 56.5 108.6^(a) 162.4 115.4  78.2 88.3^(a)  66.7^(a) 84.4  46.0  56.4  86.4  83.2  60.0 128.2  73.8^(a)22.4 28 (n = 3) 47.3  63.9  56.2 100.2 107.6^(a)  93.5 105.0 64.3  46.5 84.9  91.5  67.6^(a) 101.7  72.8^(a)  54.9 16.0 31 (n = 3) 94.5 48.5^(a)  54.8  42.2^(a)  77.5^(a)  85.7  81.4 61.0  42.8^(a)  76.8^(a) 36.2^(a) 108.4  60.0^(a)  96.9^(a) 182.4^(a)  3.8 ^(a)n = 2; ^(b)n = 6Controls used: Stimulation buffer (n = 18): 0%; Stimulation buffer andforskolin solution (n = 18): 100% DMAGO (1 μM) (n = 18): 5.5%; Morphine(1 μM) (n = 18): 6.6%

Example 14

Effects of Compound 33 on Forskolin-Stimulated Camp Responses in HEK-293Cells Transfected with μ-Opioid Receptors

The assay described in Example 5 was repeated with Compound 33. Theresults are shown in FIG. 15 expressed as a % forskolin-stimulatedcyclic AMP response. This compound produced dose dependent inhibition offorskolin-stimulated cyclic AMP formation in HEK293 cells transfectedwith MOP receptors in the μm to mm range.

Example 15

NO Release Profiles of NO Donors in a 1 and 6 Hour Assay

The assay described in Example 9 was repeated with Compounds 1, 6, 16,22, 28, 31 and 33 with NOx analysis at 1 hour and 6 hours. The resultsare shown in Table 5.

TABLE 5 NOx release Profiles over 1 and 6 hours 1 Hour 6 Hours CompoundTotal NOx (μM) Total NOx (μM) none 0.86 0.310 vehicle 0.42 0.53 1 2.082.52 1 2.19 2.52 6 0.31 0.53 16 1.08 1.19 22 1.63 2.52 28 8.94 10.04 318.60 12.92 33 13.14 67.69

Example 16

Anti-Allodynic Efficacy and Potency of NO Donor Compound 33

In rats confirmed to be hyporesponsive to the anti-allodynic effects ofsingle s.c. bolus doses of morphine at 2661 nmol/kg (FIG. 16A), singles.c. bolus doses of Compound 33 (80, 120, 800, 1200 nmol/kg; n=6-10 perdose) were administered and relief from mechanical allodynia assessed byanalysing paw withdrawal thresholds as described in Example 1.

Compound 33 produced significant relief of mechanical allodynia at 120,800 and 1200 nmol/kg. Peak levels of anti-allodynia were observed atabout 0.75-1 hour post-dosing and the corresponding duration was 2-3hours. In contrast, 80 nmol/kg of Compound 33 (n=6) producedinsignificant relief, analogous to vehicle (n=7) as shown in FIG. 16B.

What is claimed is:
 1. A compound of formula (I):

wherein one of R₁ and R₂ is selected from C₁₋₃alkyl; and the other of R₁and R₂ is selected from C₁alkylphenyl substituted with hydroxyl; ethylsubstituted with hydroxyl; and —CH₂N(ethyl)₂.
 2. The compound accordingto claim 1 wherein one of R₁ and R₂ is methyl.
 3. The compound accordingto claim 1 wherein one of R₁ and R₂ is methyl and the other isC₁alkylphenyl substituted with hydroxyl.
 4. The compound according toclaim 3 wherein the hydroxyl is substituted on the alkyl group.
 5. Thecompound according to claim 1 wherein one of R₁ and R₂ is methyl and theother is ethyl substituted with hydroxyl.
 6. The compound according toclaim 1 wherein one of R₁ and R₂ is methyl and the other is—CH₂N(ethyl)₂.